Quinoline derivatives for modulating dna methylation

ABSTRACT

Quinoline derivatives, particularly 4-anilinoquinoline derivatives, are provided. Such quinoline derivatives can be used for modulation of DNA methylation, such as effective inhibition of methylation of cytosine at the C-5 position, for example via selective inhibition of DNA methyltransferase DNMT1. Methods for synthesizing numerous 4-anilinoquinoline derivatives and for modulating DNA methylation are provided. Also provided are methods for formulating and administering these compounds or compositions to treat conditions such as cancer and hematological disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/422,067, filed Apr. 10, 2009 (allowed); which is acontinuation-in-part of U.S. application Ser. No. 11/959,181, filed Dec.18, 2007 (U.S. Pat. No. 7,790,746), which is a continuation-in-part ofU.S. application Ser. No. 11/871,762 filed Oct. 12, 2007 (abandoned),all of which applications are incorporated herein by reference in theirentireties.

BACKGROUND

1. Technical Field

The present invention relates to compounds, compositions, formulations,kits, methods of use, and manufacture of quinoline derivatives, and moreparticularly to 4-anilinoquinoline derivatives as inhibitors of DNAmethylation enzymes, modulators of DNA methylation and therapeuticagents for preventing or treating diseases associated with aberrant DNAmethylation such as cancer and hematological malignancy.

2. Description of the Related Art

Methylation at the C-5 position of cytosine residues in 5′-m⁵ CpG-3′sequences plays a major role in gene expression by the silencing ofgenes (Smith, A. F. A. Curr. Opin. Genet. Devel., 1999, 9, 657). Themajor enzyme responsible for the maintenance of these methylationpatterns during replication is the DNA methyltransferase DNMT1(Siedlecki et al., Biochem. Biophys. Res. Comm., 2003, 306, 558). DNAmethylation is the cause of a number of inherited disease syndromes, andcan also have a major role in the development of human cancer. It is themost frequent molecular change in hematopoietic neoplasms (Egger et al.,Nature, 2004, 429, 457), and is likely involved in other tumor types;for example, a percentage of patients with sporadic colorectal cancersshow methylation and silencing of the gene encoding MLH1 (Kane et al.,Cancer Res., 1997, 57, 808).

The most widely-explored inhibitors of DNMT1 are suicide inhibitors suchas azacitidine (Vidaza®) and decitabine (Dacogen®), antimetabolites thatincorporate into DNA in place of cytosine, and irreversibly trap theenzyme (Egger et al., Nature, 2004, 429, 457; Zhou et al., J. Mol.Biol., 2002, 321, 599). Both compounds are now used clinically for thetreatment of myelodysplastic syndromes and lymphoproliferative diseases,but do possess considerable toxicity (Leone et al., Clin. Immunol.,2003, 109, 89). Presumably this is due to incorporation of 5-azacytidineinto the DNA. Incorporation of decitabine into the DNA strand has ahypomethylation effect. Each class of differentiated cells has its owndistinct methylation pattern. After chromosomal duplication, in order toconserve this pattern of methylation, the 5-methylcytosine on theparental strand serves to direct methylation on the complementarydaughter DNA strand. Substituting the carbon at the 5 position of thecytosine for a nitrogen interferes with this normal process of DNAmethylation. The replacement of 5-methylcytosine with decitabine at aspecific site of methylation produces an irreversible inactivation ofDNA methyltransferase, presumably due to formation of a covalent bondbetween the enzyme and decitabine (Juttermann et al., Proc. Natl. Acad.Sci. USA, 1994, 91, 11797). By specifically inhibiting DNMT1, the enzymerequired for maintenance methylation, the aberrant methylation of thetumor suppressor genes can be prevented.

Only a few other small-molecule inhibitors of DNA methylation have beendescribed, including the psammaplin sponge metabolites (Piña et al., J.Org. Chem., 2003, 68, 3866), which are potent direct inhibitors of DNAmethyltransferases but are less effective in cellular assays (Godert etal., Bioorg. Med. Chem. Lett., 2006, 16, 3330). Other non-nucleosidedemethylating agents such as (−)-epigallocatechin-3-gallate,hydralazine, and procainamide were also shown to be far less effectivein reactivating genes than decitabine (Chuang et al., Mol. Cancer.Ther., 2005, 4, 1515).

Thus, there still exists a need to develop effective modulators of DNAmethylation which can be used in the prevention or treatment of diseasesassociated with aberrant DNA methylation such as cancer andhematological malignancy.

BRIEF SUMMARY

In one aspect, the present invention provides a compound of formula (I),or a physiologically acceptable salt or a phosphate prodrug, or acarboxylic acid or aminoacid ester prodrug thereof,

wherein G₁, G₂, G₃, and G₄ are each independently C, N, or N+ (where anR⁶-R⁹ is attached to N); G₅ and G₆ are each independently CH or N; G₇and G₈ are each independently CH, C (where an R² is attached to C), N,or N+ (where an R² is attached to N),

D₁ and D₂ are each separately CH, C (where R³ attached to C), N, or N+(where an R³ is attached to N).

R⁶, R⁷, R⁸, and R⁹ are each separately H, halogen, CF₃, OCF₃, CN,CONHR⁴, CONR⁴R⁵, SO₂Me, SO₂NHR⁴, SO₂NR⁴R⁵, NHCOR⁴, NHR⁴, NR⁴R⁵, OR⁴,NO₂, or CH₂R⁴, wherein R⁴ and R⁵ are each independently H, lower C₁-C₆alkyl or cycloalkyl optionally substituted with amino, hydroxyl,methoxy, —CN, —COOH or SO₂NH₂ groups, or with one or more oxygen, sulfuror nitrogen atoms as part of the cycloalkyl structure which mayrepresent morpholine, pyrrolidine, piperidine, pyrrolidine,thiomorpholine, imidazole or 4-methylpiperazine, or may be substitutionof a —CH═ ring carbon by —N═,

R² and R³ are each independently H, NHR⁴, NR⁴R⁵, OR⁴, NO₂ or CH₂R⁴,wherein R⁴ and R⁵ are defined as above,

X may be H or C₁-C₆ alkyl optionally substituted with amino, hydroxyl ormethoxy groups, or with one or more oxygen or nitrogen atoms as part ofa cycloalkyl structure which may represent azetidine, pyrrolidine,piperidine, piperazine, or morpholine;

Y may be CONR⁴, NR⁴CO, O, S(O), [n=0 to 2], (CH₂)_(k) [k=1 to 6],—CH═CH—, NR⁴, or a direct link between the two aromatic rings (i.e., aC—C bond between the two aromatic rings), wherein R⁴ and R⁵ are definedas above;

o, m and p represent positions of attachment of the moiety Z;

Z may be one of the groups Q1-Q43 represented in formula (II);

wherein A is O, S(O)_(w) [w=0 to 2] or NR⁴ where R⁴ is defined as above,

G₉-G₁₃ are each independently C, N, or N+(where an R¹⁰-R¹³ is attachedto N); but at least three of G₉-G₁₃ are C; and

R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are each separately H, halogen, alkyl, CF₃,OCF₃, CN, CONHR⁴, CONR⁴R⁵, SO₂Me, SO₂NHR⁴, SO₂NR⁴R⁵, NHCOR⁴, NHR⁴,NR⁴R⁵, OR⁴, NO₂ or CH₂R⁴, wherein R⁴ and R⁵ are as defined above.

It is appreciated that compounds of Formula (I) may occur in differentgeometric and enantiomeric forms, and that both pure forms and mixturesof these separate isomers are included, and any physiologicallyfunctional salt derivatives or phosphate or carboxylic acid or aminoacid ester prodrugs thereof.

In another aspect, the invention provides a compound of formula (III),

or a pharmaceutically acceptable salt, a phosphate prodrug, or acarboxylic acid or amino acid ester prodrug thereof, wherein

R⁶, R⁷, R⁸, and R⁹ are each separately H, halogen, CF₃, OCF₃, CN,CONHR⁴, CONR⁴R⁵, SO₂Me, SO₂NHR⁴, SO₂NR⁴R⁵, NHCOR⁴, NHR⁴, NR⁴R⁵, OR⁴, NO₂or CH₂R⁴, wherein R⁴ and R⁵ are each independently H, C₁-C₆ alkyl, orcycloalkyl that is optionally substituted with one or more amino,hydroxyl, methoxy, —CN, —COOH or —SO₂NH₂ groups;

X is H, or C₁-C₆ alkyl that is optionally substituted with one or moreamino, hydroxyl or methoxy groups;

Y is CONR⁴, NR⁴CO, O, S(O), (n=0 to 2), (CH₂)_(k) (k=1 to 6), —CH═CH—,or NR⁴, where R⁴ and R⁵ are defined as above;

G₇ and G₈ are each independently CH or N,

D₁ and D₂ are each independently CH or N,

o, m and p represent positions of attachment of the moiety Z;

Z is one of the groups Q1-Q43 represented in formula (II) above,wherein,

A is O, S(O)_(W) [w=0 to 2] or NR⁴, where R⁴ is defined as above;

G₉-G₁₃ are each independently C, N, or N+(where an R¹⁰-R¹³ is attachedto N), but at least three of G₉-G₁₃ are C,

R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are each separately H, halogen, alkyl, CF₃,OCF₃, CN, CONHR⁴, CONR⁴R⁵, SO₂Me, SO₂NHR⁴, SO₂NR⁴R⁵, NHCOR⁴, NHR⁴,NR⁴R⁵, OR⁴, NO₂ or CH₂R⁴, and wherein R⁴ and R⁵ are as defined above.

In a yet another aspect the invention provides a method for treating orpreventing a disease associated with aberrant DNA methylation, such ascancer, including the step of administering a compound of formula (I) or(III) or its salts or prodrugs, wherein R²-R¹⁴, G₁-G₁₃, o, m, p, X, Y,Z, and A, are defined as above, respectively for each formula.

Preferably, the subject in need of treatment or prevention of such adisease is in need of some reduction in the function/activity of themaintenance methylase DNMT1, preferably at least 25%, more preferably atleast 50%, and most preferably at least 75%, reduction of suchfunction/activity. It is envisaged that at least 25% reduction in DNAmethylase activity may be beneficial.

The method may also include co-administering one or more therapeuticagents and/or therapies. It is preferred that the method of therapyfurther includes the step of administering one or more chemotherapeuticor biologic therapeutic agents to, or radiating the subject before,during or after the administration of the compound of formula (I) or(III) or its salts or prodrugs as defined above.

While these compounds will typically be used in disease prevention ortreatment of human subjects, they can be used to treat or preventdiseases in other mammals, such as warm blooded animal subjects (e.g.,other primates, farm animals such as cattle, and sports animals and petssuch as horses, dogs, and cats).

In yet another aspect the present invention provides a pharmaceuticalcomposition comprising a compound of formula (I) or (III) or its saltsor prodrugs as defined above, and a pharmaceutically acceptableexcipient, adjuvant, carrier, buffer or stabiliser.

In yet another aspect the present invention provides a method forsynthesizing or manufacturing a compound of formula (I) or (III) or itssalts or prodrugs as defined above. Examples of such a method areillustrated in Schemes 1-6 below.

In yet another aspect the present invention provides a method formanufacture of a medicament comprising a compound of formula (I) or(III) or its salts or prodrugs as defined above for administration to asubject.

Examples of some preferred compound of Formula (I) is listed in Table 1below, wherein R², R³ and R⁴ are H, and A is NH.

TABLE 1 SOME PREFERRED COMPOUNDS OF THE PRESENT INVENTION. (I)

(R², R³ and R⁴ are H, D₁ and D₂ are CH, X is H, and A is NH) R⁶-R⁹* Yattach Z G₅ G₆ G₁ G₂ G₃ G₄ G₇ G₈ — CONH m Q1 CH CH C C C C CH CH — CONHm Q1 N CH C C C C CH CH — CONH m Q1 CH N C C C C CH CH — CONH m Q1 CH CHN C C C CH CH — CONH m Q1 CH CH C N C C CH CH — CONH m Q1 CH CH C C N CCH CH — CONH m Q1 CH CH C C C N CH CH — CONH m Q1 CH CH C C C C N CH —CONH m Q1 CH CH C C C C CH N — CONH p Q1 CH CH C C C C CH CH — CONH p Q1N CH C C C C CH CH — CONH p Q1 CH N C C C C CH CH — CONH p Ql CH CH N CC C CH CH — CONH p Q1 CH CH C N C C CH CH — CONH p Ql CH CH C C N C CHCH — CONH p Q1 CH CH C C C N CH CH — CONH p Q1 CH CH C C C C N CH — CONHp Q1 CH CH C C C C CH N — NHCO m Q1 CH CH C C C C CH CH — NHCO p Ql CHCH C C C C CH CH — CONH p Q2 CH CH C C C C CH CH — CONH p Q2 N CH C C CC CH CH — CONH p Q2 CH N C C C C CH CH — CONH p Q2 CH CH N C C C CH CH —CONH p Q2 CH CH C N C C CH CH — CONH p Q2 CH CH C C N C CH CH — CONH pQ2 CH CH C C C N CH CH — CONH p Q2 CH CH C C C C N CH — CONH p Q2 CH CHC C C C CH N — CONH p Q3 CH CH C C C C CH CH — CONH p Q4 CH CH C C C CCH CH — CONH p Q4 N CH C C C C CH CH — CONH p Q4 CH N C C C C CH CH —CONH p Q4 CH CH N C C C CH CH — CONH p Q4 CH CH C N C C CH CH — CONH pQ4 CH CH C C N C CH CH — CONH p Q4 CH CH C C C N CH CH — CONH p Q4 CH CHC C C C N CH — CONH p Q4 CH CH C C C C CH N — CONH m Q2 CH CH C C C C CHCH — CONH m Q2 N CH C C C C CH CH — CONH m Q2 CH N C C C C CH CH — CONHm Q2 CH CH N C C C CH CH — CONH m Q2 CH CH C N C C CH CH — CONH m Q2 CHCH C C N C CH CH — CONH m Q2 CH CH C C C N CH CH — CONH m Q2 CH CH C C CC N CH — CONH m Q2 CH CH C C C C CH N — CONH m Q3 CH CH C C C C CH CH —CONH m Q4 CH CH C C C C CH CH — CONH m Q4 N CH C C C C CH CH — CONH m Q4CH N C C C C CH CH — CONH m Q4 CH CH N C C C CH CH — CONH m Q4 CH CH C NC C CH CH — CONH m Q4 CH CH C C N C CH CH — CONH m Q4 CH CH C C C N CHCH — CONH m Q2 CH CH C C C C N CH — CONH m Q2 CH CH C C C C CH N R⁷ =NO₂ CONH m Q2 CH CH C C C C CH CH R⁷ = NO₂ CONH p Q2 CH CH C C C C CH CHR⁷ = NO₂ NHCO p Q2 CH CH C C C C CH CH R⁷ = NH₂ CONH m Q1 CH CH C C C CCH CH R⁷ = NH₂ CONH p Q1 CH CH C C C C CH CH R⁷ = NH₂ NHCO m Q1 CH CH CC C C CH CH R⁷ = NH₂ NHCO p Q1 CH CH C C C C CH CH R⁷ = NH₂ CONH m Q2 CHCH C C C C CH CH R⁷ = NH₂ CONH p Q2 CH CH C C C C CH CH R⁷ = NH₂ NHCO mQ2 CH CH C C C C CH CH R⁷ = NH₂ NHCO p Q2 CH CH C C C C CH CH R⁷ = NH₂CONH m Q4 CH CH C C C C CH CH R⁷ = NH₂ CONH p Q4 CH CH C C C C CH CH R⁷= NH₂ NHCO m Q4 CH CH C C C C CH CH R⁷ = NH₂ NHCO p Q4 CH CH C C C C CHCH R⁷ = NH₂ CONH m Q2 N CH C C C C CH CH R⁷ = NH₂ CONH p Q2 N CH C C C CCH CH R⁷ = NH₂ NHCO m Q2 N CH C C C C CH CH R⁷ = NH₂ NHCO p Q2 N CH C CC C CH CH R⁷ = NH₂ CONH m Q2 CH N C C C C CH CH R⁷ = NH₂ CONH p Q2 CH NC C C C CH CH R⁷ = NH₂ NHCO m Q2 CH N C C C C CH CH R⁷ = NH₂ NHCO p Q2CH N C C C C CH CH R⁷ = NH₂ CONH m Q2 CH CH N C C C CH CH R⁷ = NH₂ CONHp Q2 CH CH N C C C CH CH R⁷ = NH₂ NHCO m Q2 CH CH N C C C CH CH R⁷ = NH₂NHCO p Q2 CH CH N C C C CH CH R⁷ = NH₂ CONH m Q2 CH CH C C N C CH CH R⁷= NH₂ CONH p Q2 CH CH C C N C CH CH R⁷ = NH₂ NHCO m Q2 CH CH C C N C CHCH R⁷ = NH₂ NHCO p Q2 CH CH C C N C CH CH R⁷ = NH₂ CONH m Q2 CH CH C C CN CH CH R⁷ = NH₂ CONH p Q2 CH CH C C C N CH CH R⁷ = NH₂ NHCO m Q2 CH CHC C C N CH CH R⁷ = NH₂ NHCO p Q2 CH CH C C C N CH CH R⁷ = NH₂ CONH m Q2CH CH C C C C N CH R⁷ = NH₂ CONH p Q2 CH CH C C C C N CH R⁷ = NH₂ NHCO mQ2 CH CH C C C C N CH R⁷ = NH₂ NHCO p Q2 CH CH C C C C N CH R⁷ = NH₂CONH m Q2 CH CH C C C C CH N R⁷ = NH₂ CONH p Q2 CH CH C C C C CH N R⁷ =NH₂ NHCO m Q2 CH CH C C C C CH N R⁷ = NH₂ NHCO p Q2 CH CH C C C C CH NR⁷ = NH₂ CONH m Q3 CH CH C C C C CH CH R⁷ = NH₂ CONH p Q3 CH CH C C C CCH CH R⁷ = NH₂ NHCO m Q3 CH CH C C C C CH CH R⁷ = NH₂ NHCO p Q3 CH CH CC C C CH CH R⁷ = NMe₂ CONH p Q1 CH CH C C C C CH CH R⁷ = NMe₂ CONH m Q1CH CH C C C C CH CH R⁷ = NMe₂ NHCO p Q1 CH CH C C C C CH CH R⁷ = NMe₂NHCO m Q1 CH CH C C C C CH CH R⁷ = NMe₂ CONH p Q2 CH CH C C C C CH CH R⁷= NMe₂ CONH m Q2 CH CH C C C C CH CH R⁷ = NMe₂ NHCO p Q2 CH CH C C C CCH CH R⁷ = NMe₂ NHCO m Q2 CH CH C C C C CH CH R⁷ = NMe₂ CONH p Q3 CH CHC C C C CH CH R⁷ = NMe₂ CONH m Q3 CH CH C C C C CH CH R⁷ = NMe₂ NHCO pQ3 CH CH C C C C CH CH R⁷ = NMe₂ NHCO m Q3 CH CH C C C C CH CH R⁸ = NO₂CONH m Q1 CH CH C C C C CH CH R⁸ = NO₂ CONH p Q1 CH CH C C C C CH CH R⁸= NO₂ NHCO m Q1 CH CH C C C C CH CH R⁸ = NO₂ NHCO p Q1 CH CH C C C C CHCH R⁸ = NO₂ CONH m Q2 CH CH C C C C CH CH R⁸ = NO₂ CONH p Q2 CH CH C C CC CH CH R⁸ = NO₂ NHCO m Q2 CH CH C C C C CH CH R⁸ = NO₂ NHCO p Q2 CH CHC C C C CH CH R⁸ = NO₂ CONH m Q3 CH CH C C C C CH CH R⁸ = NO₂ CONH p Q3CH CH C C C C CH CH R⁸ = NO₂ NHCO m Q3 CH CH C C C C CH CH R⁸ = NO₂ NHCOp Q3 CH CH C C C C CH CH R⁸ = NH₂ CONH m Q1 CH CH C C C C CH CH R⁸ = NH₂CONH p Q1 CH CH C C C C CH CH R⁸ = NH₂ NHCO m Q1 CH CH C C C C CH CH R⁸= NH₂ NHCO p Q1 CH CH C C C C CH CH R⁸ = NH₂ CONH m Q2 CH CH C C C C CHCH R⁸ = NH₂ CONH p Q2 CH CH C C C C CH CH R⁸ = NH₂ NHCO m Q2 CH CH C C CC CH CH R⁸ = NH₂ NHCO p Q2 CH CH C C C C CH CH R⁸ = NH₂ CONH m Q2 N CH CC C C CH CH R⁸ = NH₂ CONH p Q2 N CH C C C C CH CH R⁸ = NH₂ NHCO m Q2 NCH C C C C CH CH R⁸ = NH₂ NHCO p Q2 N CH C C C C CH CH R⁸ = NH₂ CONH mQ2 CH N C C C C CH CH R⁸ = NH₂ CONH p Q2 CH N C C C C CH CH R⁸ = NH₂NHCO m Q2 CH N C C C C CH CH R⁸ = NH₂ NHCO p Q2 CH N C C C C CH CH R⁸ =NH₂ CONH m Q2 CH CH N C C C CH CH R⁸ = NH₂ CONH p Q2 CH CH N C C C CH CHR⁸ = NH₂ NHCO m Q2 CH CH N C C C CH CH R⁸ = NH₂ NHCO p Q2 CH CH N C C CCH CH R⁸ = NH₂ CONH m Q2 CH CH C N C C CH CH R⁸ = NH₂ CONH p Q2 CH CH CN C C CH CH R⁸ = NH₂ NHCO m Q2 CH CH C N C C CH CH R⁸ = NH₂ NHCO p Q2 CHCH C N C C CH CH R⁸ = NH₂ CONH m Q2 CH CH C C C N CH CH R⁸ = NH₂ CONH pQ2 CH CH C C C N CH CH R⁸ = NH₂ NHCO m Q2 CH CH C C C N CH CH R⁸ = NH₂NHCO p Q2 CH CH C C C N CH CH R⁸ = NH₂ CONH m Q2 CH CH C C C C N CH R⁸ =NH₂ CONH p Q2 CH CH C C C C N CH R⁸ = NH₂ NHCO m Q2 CH CH C C C C N CHR⁸ = NH₂ NHCO p Q2 CH CH C C C C N CH R⁸ = NH₂ CONH m Q2 CH CH C C C CCH N R⁸ = NH₂ CONH p Q2 CH CH C C C C CH N R⁸ = NH₂ NHCO m Q2 CH CH C CC C CH N R⁸ = NH₂ NHCO p Q2 CH CH C C C C CH N R⁸ = NH₂ CONH m Q2 CH N CC C C CH N R⁸ = NH₂ CONH p Q2 CH N C C C C CH N R⁸ = NH₂ NHCO m Q2 CH NC C C C CH N R⁸ = NH₂ NHCO p Q2 CH N C C C C CH N R⁸ = NH₂ CONH m Q3 CHCH N C C C CH CH R⁸ = NH₂ CONH p Q3 CH CH N C C C CH CH R⁸ = NH₂ NHCO mQ3 CH CH N C C C CH CH R⁸ = NH₂ NHCO p Q3 CH CH N C C C CH CH R⁸ = NMe₂CONH m Q1 CH CH C C C C CH CH R⁸ = NMe₂ CONH p Q1 CH CH C C C C CH CH R⁸= NMe₂ CONH m Q2 CH CH C C C C CH CH R⁸ = NMe₂ CONH p Q2 CH CH C C C CCH CH R⁸ = NMe₂ CONH m Q3 CH CH C C C C CH CH R⁸ = NMe₂ CONH p Q3 CH CHC C C C CH CH R⁸ = NMe₂ NHCO m Q1 CH CH C C C C CH CH R⁸ = NMe₂ NHCO pQ1 CH CH C C C C CH CH R⁸ = NMe₂ NHCO m Q2 CH CH C C C C CH CH R⁸ = NMe₂NHCO p Q2 CH CH C C C C CH CH R⁸ = NMe₂ NHCO m Q3 CH CH C C C C CH CH R⁸= NMe₂ NHCO p Q3 CH CH C C C C CH CH *If not specified, R⁶-R⁹ = H

It is to be recognized that certain compounds of the present inventionmay exist in one or more different enantiomeric or diastereomeric forms.It is to be understood that the enantiomeric or diastereomeric forms areincluded in the above aspects of the invention.

The term pharmacologically acceptable salt used throughout thespecification is to be taken as meaning any acid or base derived saltformed from hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic,malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic,methanesulfonic, isoethonic acids and the like and potassium carbonatesodium or potassium hydroxide ammonia, triethylamine, triethanolamineand the like.

In some embodiments of the invention, the salt of the compound offormula (I) or (III) is formed with an acid selected from the groupconsisting of hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, carboxylic, sulfonic, sulfo or phospho acids, aceticacid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid,glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid,2-acetoxybenzoic acid, embonic acid, nicotinic acid, isonicotinic acid,amino acid, glutamic acid, aspartic acid, phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid, and ascorbic acid. Insome embodiments, the salt is a sodium, calcium, lithium, potassium,ammonium, or trialkylammonium salt.

In some preferred embodiments, Z is Q1, Q2, Q3, Q4, Q9, or Q10. In somepreferred embodiments, the compound is a structure of formula (III), R⁶,R⁷, R⁸, and R⁹ are each H; X is H; Y is COHN or NHCO; Z is Q2 or Q9; Ais NH, and Z is attached at the m or p position. In some preferredembodiments, the compound is a structure of formula (III), R⁶, R⁷, R⁸,and R⁹ are each H; X is H; Y is CONH, Z is Q2; A is NH; and Z isattached at the p position. In some preferred embodiments, the compoundis a structure of formula (III), R⁶, R⁷, R⁸, and R⁹ are each H; X is H;Y is CONH; Z is Q9; A is NH; and Z is attached at the p position. Insome preferred embodiments, the compound is a structure of formula(III), R⁶, R⁷, R⁸, and R⁹ are each H; X is H; Y is CONN or NHCO; Z isQ2; A is NH, and Z is attached at the m position. In some preferredembodiments, the compound is a structure of formula (III), R⁶, R⁸, andR⁹ are each H; R⁷ is NMe₂; X is H; Y is CONH or NHCO; Z is Q2; A is NH,and Z is attached at the m position. In some preferred embodiments, thecompound is a structure of formula (III), R⁶, R⁸, and R⁹ are each H; R⁷is Cl; X is H; Y is CONH; Z is Q2; A is NH, and Z is attached at the pposition. In some preferred embodiments, the compound is a structure offormula (III), R⁶, R⁷, R⁸, and R⁹ are separately H, F, or Cl. In someembodiments, Z is Q9, Q39 or Q42. In other more specific embodiments,the compound is a structure of formula (III), R⁶, R⁷, R⁸, and R⁹ areeach H; X is H; Y is CONH or NHCO; Z is Q9, Q39 or Q42; A is S; w is 0;and Z is attached at the m or p position. In other specific embodiments,the compound is a structure of formula (III), R⁶, R⁷, R⁸, and R⁹ areeach H; X is H; Y is CONH, Z is Q9; A is S; w is 0; and Z is attached atthe p position. In other specific embodiments, the compound is astructure of formula (III), R⁶, R⁷, R⁸, and R⁹ are each H; X is H; Y isCONH; Z is Q39; A is S; w is 0; and Z is attached at the p position. Inyet other specific embodiments, the compound is a structure of formula(III), R⁶, R⁷, R⁸, and R⁹ are each H; X is H; Y is CONH; Z is Q42; A isS; w is 0; and Z is attached at the p position.

One aspect of the invention is a pharmaceutical composition comprising acompound, salt, or prodrug of formula (I) or (III) or its salts orprodrugs as defined above, and a pharmaceutically-acceptable carrier. Insome embodiments of the pharmaceutical composition, the compound, salt,or prodrug is in solid form. In some embodiments the pharmaceuticalcomposition is in an oral dosage form. In some embodiments thepharmaceutical composition is in an injectable dosage form. In someembodiments the pharmaceutical composition is in a topical dosage form.

One aspect of the invention is a method for inhibiting DNA methylationin a cell, comprising: contacting the cell with the compound of formula(I) or (III) or its salts or prodrugs as defined above, such that DNAmethylation activity of the cell is inhibited.

One aspect of the invention is a method for inhibiting DNA methylationin a cell, comprising: contacting the cell with the compound of formula(I) or (III) or its salts or prodrugs as defined above, such that DNAmethytransferase activity in the cell is inhibited. In some embodiments,the activity of DNA methyltransferase activity is inhibited viadegradation of DNA methyltransferase DNMT1. In some embodiments, thestep of contacting includes contacting the cell with a biologicallyeffective amount of the compound of formula (I) or (III) or its salts orprodrugs as defined above, such that at least 50% of the activity of DNAmethyltransferase DNMT1 in the cell is inhibited. In some embodiments,the step of contacting includes contacting the cell with a biologicallyeffective amount of the compound of formula (I) or (III) or its salts orprodrugs as defined above, such that at least 25% of the activity of DNAmethyltransferase DNMT1 in the cell is inhibited.

One aspect of the invention is a method for restoring activity of a DNAmethylation-suppressed gene in a cell, comprising: contacting a cellwith a biologically effective amount of the compound of formula (I) or(III) or its salts or prodrugs as defined above, such that activity ofthe DNA methylation-suppressed gene is elevated by at least 25% relativeto that in the absence of the compound, salt, or prodrug. In someembodiments, the step of contacting includes contacting the cell with abiologically effective amount of the compound of formula (I) or (III) orits salts or prodrugs as defined above, such that transcriptionalactivity or levels of transcript of the DNA-methylation-suppressed geneis elevated by at least 25%. In some embodiments, the DNAmethylation-suppressed gene is selected from the group consisting of14-3-3 Sigma, ABL1 (P1), ABO, APC, AR (Androgen Receptor), BLT1(Leukotriene B4 Receptor), BRCA1, CALCA (Calcitonin), CASP8 (CASPASE 8),Caveolin 1, CD44, CFTR, COX2, CSPG2 (Versican), CX26 (Connexin 26),Cyclin A1, DBCCR1, ECAD (E-cadherin), Endothelin Receptor B, EPHA3, EPO(Erythropoietin), ER (Estrogen Receptor), FHIT, GPC3 (Glypican 3),GST-pi, H19, H-Cadherin (CDH13), γ-globin, HIC1, hMLH1, HOXA5, IGF2(Insulin-Like Growth Factor II), IGFBP7, IRF7, LKB1, LRP-2 (Megalin),MDGI (Mammary-derived growth inhibitor), MDR1, MDR3 (PGY3), MGMT (O6methyl guanine methyl transferase), MUC2, MYOD1, N33, NEP (NeutralEndopeptidase 24.1)/CALLA, NIS (sodium-iodide symporter gene), P14/ARF,P15 (CDKN2B), P16 (CDKN2A), P27KIPh1, p57 KIP2, PAX6, PgR (ProgesteroneReceptor), RAR-Beta2, RASSF1, RB1 (Retinoblastoma), TERT, TESTIN,TGFBRI, THBS1 (Thrombospondin-1), TIMP3, TLS3 (T-Plastin), Urokinase(uPA), VHL (Von-Hippell Lindau), WT1, and ZO2 (Zona Occludens 2). Thewebsite of M. D. Anderson Cancer Center provides detailed information onthese tumor suppressor genes. See the website atwww.mdanderson.org/departments/methylation/dIndex.cfm?pn=D02B3250-57D7-4F61-88358636A8073A08,which is herein incorporated by reference.

One aspect of the invention is a method for treating a patient sufferingfrom a disease associated with aberrant DNA methylation, comprising:administering to the patient a pharmaceutical composition comprising atherapeutically-effective amount of the compound of formula (I) or (III)or its salts or prodrugs as defined above and apharmaceutically-acceptable carrier. In some embodiments, thepharmaceutical composition is administered orally, parenterally,topically, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery, subcutaneously, intraadiposally, intraarticularly,or intrathecally. In some embodiments, the pharmaceutical composition isadministered orally. In some embodiments, the method further involvesadministering to the patient a second therapeutic agent in combinationwith the pharmaceutical composition. In some embodiments, the secondtherapeutic agent is decitabine or azacitidine. In some embodiments thesecond therapeutic agent is selected from the group consisting ofhistone deacylase inhibitors, antibiotic agents, alkylating agents,retinoids, hormonal agents, plant-derived agents, biologic agents,interleukins, interferons, cytokines, immuno-modulating agents, andmonoclonal antibodies. In some embodiments, the histone deacylaseinhibitor is selected from the group consisting of trichostatin A,suberoylanilide hydroxamic acid, oxamflatin, suberic bishydroxamic acid,m-carboxy-cinnamic acid bishydroxamic acid, pyroxamide, trapoxin A,apicidin, depsipeptide,N-(2-amimophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide,butyric acid, phenylbutyrate and arginine butyrate.

In some embodiments, the disease associated with aberrant DNAmethylation is selected from the group consisting of hematologicaldisorders, benign tumor and cancer. In some embodiments thehematological disorder is selected from the group consisting of acutemyeloid leukemia, acute promyelocytic leukemia, acute lymphoblasticleukemia, chronic myelogenous leukemia, myelodysplastic syndromes, andsickle cell anemia. In some embodiments, the disease is cancer and isselected from group consisting of breast cancer, skin cancer, bonecancer, prostate cancer, liver cancer, lung cancer, non-small cell lungcancer, brain cancer, cancer of the larynx, gall bladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, and kidney cancer, basal cell carcinoma,squamous cell carcinoma of both ulcerating and papillary type,metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulumcell sarcoma, myeloma, giant cell tumor, small-cell lung tumor,gallstones, islet cell tumor, primary brain tumor, acute and chroniclymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicsarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multi form a, leukemias, lymphomas,malignant melanomas, and epidermoid carcinomas.

In yet another aspect the present invention provides a method forinhibiting DNA methylation in a cell, comprising: contacting the cellwith the compound of formula (I) or (III) or its salts or prodrugs asdefined above, such that DNA methylation activity of the cell isinhibited, preferably by at least 25%, more preferably by at least 50%,relative to the DNA methylation activity prior to the administration ofthe compound.

In yet another aspect the present invention provides a method forselectively inhibiting activity of DNA methyltransferase DNMT1 in acell, comprising: contacting a cell with the compound of the presentinvention, such that activity of DNA methyltransferase DNMT1 in the cellis inhibited more than that of DNA methyltransferase DNMT3a or DNMT3b.

According to the method, the activity of DNA methyltransferase DNMT1 isinhibited via degradation of DNA methyltransferase DNMT1.

According to the method, the step of contacting includes contacting thecell with a biologically effective amount of the compound of presentinvention, such that at least 50% of the activity of DNAmethyltransferase DNMT1 in the cell is inhibited.

According to the method, the step of contacting includes contacting thecell with a biologically effective amount of the compound of the presentinvention, such that at least 25% of the activity of DNAmethyltransferase DNMT1 in the cell is inhibited.

In yet another aspect the present invention provides a method forrestoring activity of a DNA methylation-suppressed gene in a cell,comprising: contacting a cell with a biologically effective amount ofthe compound of the present invention, such that activity of the DNAmethylation-suppressed gene is elevated by at least 25%, preferably atleast 50%, relative to that in the absence of the compound. The activityof the DNA methylation-suppressed gene includes but is not limited totranscriptional activity of the methylation-suppressed gene.

In yet another aspect the present invention provides a method fortreating a patient suffering from a disease associated with aberrant DNAmethylation, comprising: administering to the patient a pharmaceuticalcomposition comprising a therapeutically-effective amount of thecompound of formula (I) or (III) or its salts or prodrugs as definedabove and a pharmaceutically-acceptable carrier.

According to the method, the pharmaceutical composition is administeredorally, parenterally, topically, intraperitoneally, intravenously,intraarterially, transdermally, sublingually, intramuscularly, rectally,transbuccally, intranasally, liposomally, via inhalation, vaginally,intraoccularly, via local delivery, subcutaneously, intraadiposally,intraarticularly, or intrathecally.

According to the method, the method further comprises: administering tothe patient a second therapeutic agent in combination with thepharmaceutical composition.

According to the method, the second therapeutic agent may be decitabineor azacitidine.

According to the method, the second therapeutic agent may be selectedfrom the group consisting of histone deacylase inhibitor, antibioticagents, alkylating agents, retinoids, hormonal agents, plant-derivedagents, biologic agents, interleukins, interferons, cytokines,immuno-modulating agents, and monoclonal antibodies.

According to the method, the disease associated with aberrant DNAmethylation is selected from the group consisting of hematologicaldisorders, benign tumor and cancer.

Examples of cancer include but are not limited to acute myeloidleukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia,chronic myelogenous leukemia, myelodysplastic syndromes, and sickle cellanemia.

Examples of the hematological disorder include but are not limited tobreast cancer, skin cancer, bone cancer, prostate cancer, liver cancer,lung cancer, non-small cell lung cancer, brain cancer, cancer of thelarynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal,neural tissue, head and neck, colon, stomach, bronchi, and kidneycancer, basal cell carcinoma, squamous cell carcinoma of both ulceratingand papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing'ssarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-celllung tumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicsarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, and epidermoid carcinomas.

Further aspects of the present invention will become apparent from thefollowing description given by way of example only and with reference tothe accompanying synthetic schemes.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates the increase of RNA expression levels of p16 byrepresentative compounds of this invention at different concentrations.

DETAILED DESCRIPTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The present invention provides compounds, compositions, formulations,kits, methods of use, and manufacture of quinoline derivatives, and moreparticularly to 4-anilinoquinoline derivatives as inhibitors of DNAmethylation enzymes, modulators of DNA methylation and therapeuticagents for preventing or treating diseases associated with aberrant DNAmethylation such as cancer and hematological malignancy.

We have unexpectedly found that a series of 4-anilinoquinolines (e.g.,compounds of Formula (I) or Formula (III)) specifically inhibit thecellular functions of the DNA methyltransferase DNMT1 via selectivedegradation of DNMT1. In contrast, previously 4-anilinoquinoliniumbis-quaternary salts have been shown to bind in the DNA minor groove(Leupin et al., Biochemistry, 1986, 25, 5902; Squire et al., NucleicAcids Res., 1997, 25, 4072). These have been shown to be cytotoxic, andwere originally developed as anticancer drugs (Atwell & Cain, J. Med.Chem., 1973, 16, 673; Denny et al., J. Med. Chem., 1979, 22, 134), butthe example (NSC 176319) considered for clinical trial was found to betoo toxic (Plowman & Adamson, Pharmacol., 1978, 17, 61).

While not wishing to be bound to the exact mechanism of action of thecompounds of present invention, we believe that the inventive4-anilinoquinoline derivatives, in comparison with 4-anilinoquinoliniumbis-quaternary salts in the art, should be more specific in theinhibition of DNMT1 and/or in the inhibition of DNA methylation in thecell due to their fewer charged substitutes or the replacement of thequaternary quinolinium and/or the pyridinium functional groups with oneswith lower pKa values. Thus, the compounds of present invention shouldhave reduced cytotoxicity and more specifically modulate DNA methylationactivity in the cell.

1. Methods for Preparing Compounds of the Invention

The compounds of the invention can be prepared by the methods shown inSchemes 1-6 below, and given in the detailed examples.

In Scheme 1, compounds where Y is CONN and Z is Q1 in Formula (I) can beprepared by reaction of 4-(pyridyl)pyridinium with nitroanilines (2),reduction of these with H2/Pd to amines (3), then reaction of these with4-nitrobenzoic acid to gave amides (6), which can be quaternised (forexample, with MeOTs) to give pyridinium compounds (7). Reduction ofthese with Fe dust/HCl gives amines (8), which can be coupled with4-chloroquinolines, and if necessary further processed, to givecompounds of Formula (I).

In Scheme 2, compounds where Y is NHCO and Z is Q1 in Formula (I) can beprepared by reacting N-acetylaminobenzoic acids (9) with 4-nitroanilineusing coupling reagents such as EDCI or CDI to give amides (10), whichcan be hydrolysed with dilute HCl in 1,4-dioxane to amines (11). Thesecan be reacted with 4-pyridyl pyridiniumchloride to give amines (12).Quaternisation of these as above (for example, with MeOTs) givespyridinium compounds (13), which can be reduced to amines (14) and thencoupled with 4-chloroquinolines as before, and if necessary furtherprocessed, to give compounds of Formula (I).

In Scheme 3, compounds where Y is NHCO and Z is Q3 in Formula (I) can beprepared by coupling of 4-chloroquinolines (15) and 4-acetylaminoaniline(16) to give anilinoquinolines (17), and deblocking these to giveanilinoquinolines (18). Reaction of acetylbenzoic acids (19) withaminoguanidine gives guanylhydrazones (20), and these are coupled withanilinoquinolines (18) using EDCI or some other coupling reagent to givecompounds of Formula I.

In Scheme 4, compounds where Y is NHCO and Z is Q2 in Formula (I) can beprepared by coupling 4-anilinoquinolines (18) with N-acetylbenzoic acids(21) to give amides (22), which are hydrolysed to amines (23) thenreacted with 2-amino-6-chloro-4-methylpyrimidine to give compounds ofFormula I.

In Scheme 5, compounds where Y is CONH and Z is Q3 in Formula (I) can beprepared by reaction of nitroacetophenones (24) with aminoguanidine,followed by reduction of the product guanylhydrazones with Fe/HCl (25)to give the amines (26). These are reacted with 4-nitrobenzoyl chloride,and the product amides (27) are again reduced to the amines (28).Coupling of these with 4-chloroquinolines then gives the compounds ofFormula I.

In Scheme 6, compounds where Y is CONH and Z is Q2 in Formula (I) can beprepared by coupling nitroanilines (2) with2-amino-6-chloro-4-methylpyrimidine, and reduction of the resultingproducts (27) with Fe/HCl to give amines (28). Coupling of these with4-nitrobenzoyl chloride, and reduction of the products (29) as beforegave the amines (30). These were coupled with 4-chloroquinolines asabove to give compounds of formula (I).

Compounds of Formula (I), where A is S, can be prepared according toScheme 7. An appropriately substituted thiol where X is either NO₂ (31)or NH₂ (32) can be reacted with a chloro Q derivative (33), wherein 33represents any of Q1-Q43 wherein A (or the bond at the point ofconnection in the case of Q3) is replaced with Cl, to obtain compoundsof structure 34 (X═NO₂) or 35 (X═NH₂). Compounds of structure 34 can bereduced with an appropriate reducing agent, for example iron andhydrochloric acid, to produce compounds of structure 35. Reaction of anappropriately substituted benzoyl chloride (36) with compounds ofstructure 35 yields compounds of structure 37. Reduction of compounds ofstructure 37 with an appropriate reducing agent, for example iron andhydrochloric acid, produces compounds of structure 38. Reaction ofcompounds of structure 38 with an appropriately substituted4-chloroquinoline (39) then yields compounds of structure (I) wherein Qrepresents any of Q1-Q43 and w is 0. Further oxidation with anappropriate oxidant, for example meta-chloroperbenzoic acid), yieldscompounds of structure (I) wherein Q represents and of Q1-Q43 and w is 1or 2.

The quinoline derivatives of the present invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of thequinoline derivatives of the invention may be prepared by forming one ormore ester bond with any of the hydroxyl groups in the compounds usingan organic compound containing a carboxyl group, or as SATE[(S-acetyl-2-thioethyl)phosphate) derivatives according to the methodsdisclosed in WO 93/2451, or in WO 94/26764 and U.S. Pat. No. 5,770,713.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Thus, 4-anilinoquinolinium bisquaternary salts are preferably excluded.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention.

As used herein, a “pharmaceutical addition salt” includes apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These include organicor inorganic acid salts of the amines. Suitable pharmaceuticallyacceptable salts are well known to those skilled in the art and includebasic salts of a variety of inorganic and organic acids, such as, forexample, with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid or phosphoric acid; with organiccarboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamicacids, for example acetic acid, propionic acid, glycolic acid, succinicacid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid,malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid,glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinicacid or isonicotinic acid; and with amino acids, such as the 20alpha-amino acids involved in the synthesis of proteins in nature, forexample glutamic acid or aspartic acid, and also with phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

The invention also embraces isolated compounds. An isolated compoundrefers to a compound which represents at least 10%, preferably 20%, morepreferably 50% and most preferably 80% of the compound present in themixture, and exhibits a detectable (i.e., statistically significant)inhibitory activity of DNA methylation directly or indirectly whentested in biological assays such as the combined bisulfite restrictionanalysis or COBRA (Xiong, Z.; Laird, P. W. Nucleic Acids Res. 1997, 25,2532-2534), radiolabeled methyl incorporation assay (Francis, K. T.;Thompson, R. W.; Krumdieck, C. L. Am. J. Clin. Nutr. 1977, 30,2028-2032), and the DNMT assays described in Ghoshal et al (2005)11:4727-41 and in the EXAMPLE section below. Preferably, the compoundsof the present invention selectively inhibit activity of DNMT1 relativeto DNMT3a and DNMT3b.

2. Pharmaceutical Formulations of the Present Invention

According to the present invention, the compounds of the presentinvention can be formulated into pharmaceutically acceptablecompositions for treating various diseases and conditions.

The pharmaceutically-acceptable compositions of the present inventioncomprise one or more compounds of the invention in association with oneor more nontoxic, pharmaceutically-acceptable carriers and/or diluentsand/or adjuvants and/or excipients, collectively referred to herein as“carrier” materials, and if desired other active ingredients.

The compounds of the present invention are administered by any route,preferably in the form of a pharmaceutical composition adapted to such aroute, as illustrated below and are dependent on the condition beingtreated. The compounds and compositions can be, for example,administered orally, parenterally, intraperitoneally, intravenously,intraarterially, transdermally, sublingually, intramuscularly, rectally,transbuccally, intranasally, liposomally, via inhalation, vaginally,intraoccularly, via local delivery (for example by a catheter or stent),subcutaneously, intraadiposally, intraarticularly, or intrathecally.

The pharmaceutical formulation may optionally further include anexcipient added in an amount sufficient to enhance the stability of thecomposition, maintain the product in solution, or prevent side effects(e.g., potential ulceration, vascular irritation or extravasation)associated with the administration of the inventive formulation.Examples of excipients include, but are not limited to, mannitol,sorbitol, lactose, dextrose, cyclodextrin such as, α-, β-, andγ-cyclodextrin, and modified, amorphous cyclodextrin such ashydroxypropyl-, hydroxyethyl-, glucosyl-, maltosyl-, maltotriosyl-,carboxyamidomethyl-, carboxymethyl-, sulfobutylether-, anddiethylamino-substituted α, β-, and γ-cyclodextrin. Cyclodextrins suchas Encapsin® from Janssen Pharmaceuticals or equivalent may be used forthis purpose.

For oral administration, the pharmaceutical compositions can be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica, or talc;disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs may contain conventional additives such as suspending agents,emulsifying agents, non-aqueous agents, preservatives, coloring agentsand flavoring agents. Examples of additives for liquid preparationsinclude acacia, almond oil, ethyl alcohol, fractionated coconut oil,gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin,methyl cellulose, methyl or propyl para-hydroxybenzoate, propyleneglycol, sorbitol, or sorbic acid.

For topical use the compounds of the present invention can also beprepared in suitable forms to be applied to the skin, or mucus membranesof the nose and throat, and can take the form of creams, ointments,liquid sprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compounds of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery.

The pharmaceutical compositions can be administered via injection.Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

3. Methods for Administrating Inventive Compounds/Compositions

The compounds or formulations of the present invention can beadministered by any route, preferably in the form of a pharmaceuticalcomposition adapted to such a route, as illustrated below and aredependent on the condition being treated. The compounds or formulationscan be, for example, administered orally, parenterally, topically,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery (for example by catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally. The compoundsand/or compositions according to the invention may also be administeredor co-administered in slow release dosage forms.

The compounds and/or compositions of this invention may be administeredor co-administered in any conventional dosage form. Co-administration inthe context of this invention is defined to mean the administration ofmore than one therapeutic agent in the course of a coordinated treatmentto achieve an improved clinical outcome. Such co-administration may alsobe coextensive, that is, occurring during overlapping periods of time.

The inventive compound or the composition containing the inventivecompound may be administered into a host such as a patient at a dose of0.1-1000 mg/m², optionally 1-200 mg/m², optionally 1-50 mg/m²,optionally 1-40 mg/m², optionally 1-30 mg/m², optionally 1-20 mg/m², oroptionally 5-30 mg/m².

The pharmaceutical formulations may be co-administered in anyconventional form with one or more member selected from the groupcomprising infusion fluids, therapeutic compounds, nutritious fluids,anti-microbial fluids, buffering and stabilizing agents.

Patients may receive the pharmaceutical formulations intravenously. Thepreferred route of administration is by intravenous infusion.Optionally, the pharmaceutical formulations of the current invention maybe infused directly, without prior reconstitution.

Patients may be infused with the pharmaceutical formulations for 1, 2,3, 4, 5 or more hours, as a result of the enhanced stability of theformulations. Prolonged periods of infusion enable flexible schedules ofadministration of therapeutic formulations.

Alternatively or in addition, speed and volume of the infusion can beregulated according to the patient's needs. The regulation of theinfusion of the pharmaceutical formulations can be performed accordingto existing protocols.

The pharmaceutical formulations may be co-infused in any conventionalform with one or more member selected from the group comprising infusionfluids, therapeutic compounds, nutritious fluids, anti-microbial fluids,buffering and stabilizing agents. Optionally, therapeutic componentsincluding, but are not limited to, anti-neoplastic agents, alkylatingagents, agents that are members of the retinoids superfamily, antibioticagents, hormonal agents, plant-derived agents, biologic agents,interleukins, interferons, cytokines, immuno-modulating agents, andmonoclonal antibodies, may be co-infused with the inventiveformulations.

Co-infusion in the context of this invention is defined to mean theinfusion of more than one therapeutic agent in a course of coordinatedtreatment to achieve an improved clinical outcome. Such co-infusion maybe simultaneous, overlapping, or sequential. In one particular example,co-infusion of the pharmaceutical formulations and infusion fluids maybe performed through Y-type connector.

The pharmacokinetics and metabolism of intravenously administered thepharmaceutical formulations resemble the pharmacokinetics and metabolismof intravenously administered the inventive compound.

4. Combination Therapy with Inventive Pharmaceutical Compositions

The compounds or pharmaceutical formulations of the present inventionmay be used in conjunction with a cytidine analog, such as5-aza-2′-deoxycytidine (decitabine), and 5-aza-cytidine (azacitidine).

Decitabine is an antagonist of its related natural nucleoside,deoxycytidine. The only structural difference between these twocompounds is the presence of a nitrogen at position 5 of the cytosinering in decitabine as compared to a carbon at this position fordeoxycytidine.

Decitabine possesses multiple pharmacological characteristics. At amolecular level, it is S-phase dependent for incorporation into DNA. Ata cellular level, decitabine can induce cell differentiation and exerthematological toxicity.

The most prominent function of decitabine is its ability to specificallyand potently inhibit DNA methylation. As described above for methylationof cytosine in CpG islands as an example, methylation of cytosine to5-methylcytosine occurs at the level of DNA. Inside the cell, decitabineis first converted into its active form, the phosphorylated5-aza-deoxycytidine, by deoxycytidine kinase which is primarilysynthesized during the S phase of the cell cycle. The affinity ofdecitabine for the catalytical site of deoxycytidine kinase is similarto the natural substrate, deoxycytidine. After conversion to itstriphosphate form by deoxycytidine kinase, decitabine is incorporatedinto replicating DNA at a rate similar to that of the natural substrate,dCTP. Bouchard and Momparler (1983) Mol. Pharmacol. 24:109-114.

Incorporation of decitabine into the DNA strand has a hypomethylationeffect. Each class of differentiated cells has its own distinctmethylation pattern. After chromosomal duplication, in order to conservethis pattern of methylation, the 5-methylcytosine on the parental strandserves to direct methylation on the complementary daughter DNA strand.Substituting carbon at the 5 position of the cytosine for a nitrogeninterferes with this normal process of DNA methylation. The replacementof 5-methylcytosine with decitabine at a specific site of methylationproduces an irreversible inactivation of DNA methyltransferase,presumably due to formation of a covalent bond between the enzyme anddecitabine. Juttermann et al. (1994) Proc. Natl. Acad. Sci. USA91:11797-11801. By specifically inhibiting DNA methyltransferase, theenzyme required for methylation, the aberrant methylation of the tumorsuppressor genes can be prevented.

The compounds or pharmaceutical formulations of the present inventionmay be used in conjunction with inhibitors of histone deacetylase (HDAC)to further modulate transcription of genes, e.g., to reestablishtranscription of genes silenced by hypermethylation and acetylation ofhistones, in a synergistic manner.

The compounds or pharmaceutical formulations of the present inventionmay be used in conjunction with inhibitors of histone deacetylase (HDAC)to further modulate transcription of genes, e.g., to reestablishtranscription of genes silenced by hypermethylation and acetylation ofhistones, in a synergistic manner.

HDAC plays important roles in transcription silencing of genes. Theamount of acetylation on the histones is controlled by the opposingactivities of two types of enzymes, histone acetyl transferase (HATs)and histone deacetylases (HDACs). Substrates for these enzymes includee-amino groups of lysine residues located in the amino-terminal tails ofthe histones H3, H4, H2A, and H2B. These amino acid residues areacetylated by HATs and deacetylated by HDACs. With the removal of theacetyl groups from the histone lysine by HDACs, a positive charge isrestored to the lysine residue, thereby condensing the structure ofnucleosome and silencing the genes contained within. Thus, to activatethese genes silenced by deacetylase of histones, the activity of HDACsshould be inhibited. With the inhibition of HDAC, histones areacetylated and the DNA that is tightly wrapped around a deacetylatedhistone core relaxes. The opening of DNA conformation leads toexpression of specific genes.

In addition to deacetylation of histones, HDACs may also regulated geneexpression by deacetylating transcription factors, such as p53 (a tumorsuppressor gene), GATA-1, TFIIE, and TFIIF. Gu and Roeder (1997) Cell90:595-606 (p53); and Boyes et al. (1998) Nature 396:594-598 (GATA-1).HDACs also participate in cell cycle regulation, for example, bytranscription repression which is mediated by RB tumor suppressorproteins recruiting HDACs. Brehm et al. (1998) Nature 391:597-601. Thus,inhibition of HDACs should activate expression of tumor suppressor genessuch as p53 and RB and as a result promote cell growth arrest,differentiation and apoptosis induced by these genes.

As described above, aberrant transcriptional silencing of a number ofgenes, such as tumor suppressor genes, is directly related topathogenesis of cancer and other diseases. Methylation of cytosineresidues in DNA and removal of acetyl groups from histones are the twoprimary mechanisms for gene silencing. Due to methylation and/or histonedeacetylase of cancer-related genes, expression of these genes issuppressed or completely silenced. Meanwhile, expression of these genesis required for induction of growth arrest, differentiation, and/orapoptotic cell death of transformed cells. Inaction of these genes inthe transformed cells leads to uncontrolled proliferation of thesecells, which eventually results in cancer.

By combining the inventive compounds/compositions with HDAC inhibitors,genes required for induction of growth arrest, differentiation and celldeath of transformed cells can be reactivated effectively. The inventivecompounds/compositions inhibit methylation of DNA for the genes,especially in the regulatory region, thus resulting in activation oftranscription of the gene. Meanwhile, HDAC inhibitors inhibitdeacetylase of the histones in the nucleosomal core of the gene, thusresulting in net increase of the acetylation of histones, which, inturn, activates transcription of the gene. By exploiting these twocomplementary mechanisms, the combination therapy may reestablish genetranscription more effectively and, ideally, in a synergistic manner. Acombination therapy having synergistic effects should require a lessamount of each inhibitor than it being used alone, thus reducingpotential side effects associated systemic administration of highdosages of the inhibitors and improving therapeutic index.

Many anticancer agents exert their anti-cancer effects by triggeringsignal transduction cascades involving proteins encoded by these tumorsuppressor genes. With insufficient expression of these genes in cancercells, the anti-cancer effects of these anti-neoplastic agents may beseverely reduced or completely eradicated. Through reactivation orre-expression of these genes that are epigenetically silenced by DNAmethylation and histone deacetylase, the intrinsic defense mechanisms ofthe body are mobilized to combat the disease by restoration of thetumor-suppressing functions to cancer cells in response to signals sentby the anti-cancer agent administered. Such stimulation of the intrinsictumor suppressing functions of the body should lead to the requirementof lower dosage of the anticancer agent, thus resulting in a highertherapeutic index (i.e., greater efficacy and lower toxicity) of theagent.

Inhibitors of HDACs include, but are not limited to, the followingstructural classes: 1) hydroxamic acids, 2) cyclic peptides, 3)benzamides, and 4) short-chain fatty acids.

Examples of hydroxamic acids and hydroxamic acid derivatives, but arenot limited to, trichostatin A (TSA), suberoylanilide hydroxamic acid(SAHA), oxamflatin, suberic bishydroxamic acid (SBHA),m-carboxy-cinnamic acid bishydroxamic acid (CBHA), and pyroxamide. TSAwas isolated as an antifungal antibiotic (Tsuji et al (1976) J. Antibiot(Tokyo) 29:1-6) and found to be a potent inhibitor of mammalian HDAC(Yoshida et al. (1990) J. Biol. Chem. 265:17174-17179). The finding thatTSA-resistant cell lines have an altered HDAC evidences that this enzymeis an important target for TSA. Other hydroxamic acid-based HDACinhibitors, SAHA, SBHA, and CBHA are synthetic compounds that are ableto inhibit HDAC at micromolar concentration or lower in vitro or invivo. Glick et al. (1999) Cancer Res. 59:4392-4399. These hydroxamicacid-based HDAC inhibitors all possess an essential structural feature:a polar hydroxamic terminal linked through a hydrophobic methylenespacer (e.g., 6 carbon at length) to another polar site which isattached to a terminal hydrophobic moiety (e.g., benzene ring).Compounds developed having such essential features also fall within thescope of the hydroxamic acids that may be used as HDAC inhibitors.

Cyclic peptides used as HDAC inhibitors are mainly cyclic tetrapeptides.Examples of cyclic peptides include, but are not limited to, trapoxin A,apicidin and FR901228. Trapoxin A is a cyclic tetrapeptide that containsa 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) moiety. Kijima et al. (1993)J. Biol. Chem. 268:22429-22435. Apicidin is a fungal metabolite thatexhibits potent, broad-spectrum antiprotozoal activity and inhibits HDACactivity at nanomolar concentrations. Darkin-Rattray et al. (1996) Proc.Natl. Acad. Sci. USA. 93; 13143-13147. FR901228 is a depsipeptide thatis isolated from Chromobacterium violaceum, and has been shown toinhibit HDAC activity at micromolar concentrations.

Examples of benzamides include but are not limited to MS-27-275. Saitoet al. (1990) Proc. Natl. Acad. Sci. USA. 96:4592-4597. Examples ofshort-chain fatty acids include but are not limited to butyrates (e.g.,butyric acid, arginine butyrate and phenylbutyrate (PB)). Newmark et al.(1994) Cancer Lett. 78:1-5; and Carducci et al. (1997) Anticancer Res.17:3972-3973. In addition, depudecin which has been shown to inhibitHDAC at micromolar concentrations (Kwon et al. (1998) Proc. Natl. Acad.Sci. USA. 95:3356-3361) also falls within the scope of histonedeacetylase inhibitor of the present invention.

The compounds or pharmaceutical formulations of the present inventionmay also be used in conjunction with other therapeutic componentsincluding but not limiting to anti-neoplastic agents, alkylating agents,agents that are members of the retinoids superfamily, antibiotic agents,hormonal agents, plant-derived agents, biologic agents, interleukins,interferons, cytokines, immuno-modulating agents, and monoclonalantibodies.

In one embodiment, an alkylating agent is used in combination withand/or added to the inventive compound/formulation. Examples ofalkylating agents include, but are not limited to bischloroethylamines(nitrogen mustards, e.g., chlorambucil, cyclophosphamide, ifosfamide,mechlorethamine, melphalan, uracil mustard), aziridines (e.g.,thiotepa), alkyl alkone sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomustine, streptozocin), nonclassic alkylating agents(altretamine, dacarbazine, and procarbazine), platinum compounds(carboplastin and cisplatin).

In another embodiment, cisplatin, carboplatin or cyclophosphamide isused in combination with and/or added to the inventivecompound/formulation.

In another embodiment, a member of the retinoids superfamily is used incombination with and/or added to the inventive compound/formulation.Retinoids are a family of structurally and functionally relatedmolecules that are derived or related to vitamin A (all-trans-retinol).Examples of retinoid include, but are not limited to, all-trans-retinol,all-trans-retinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin)and 9-cis-retinoic acid.

In yet another embodiment, a hormonal agent is used in combination withand/or added to the inventive compound/formulation. Examples of such ahormonal agent are synthetic estrogens (e.g., diethylstibestrol),antiestrogens (e.g., tamoxifen, toremifene, fluoxymesterol andraloxifene), antiandrogens (bicalutamide, nilutamide, flutamide),aromatase inhibitors (e.g., aminoglutethimide, anastrozole andtetrazole), ketoconazole, goserelin acetate, leuprolide, megestrolacetate and mifepristone.

In yet another embodiment, a plant-derived agent is used in combinationwith and/or added to the inventive compound/formulation. Examples ofplant-derived agents include, but are not limited to, vinca alkaloids(e.g., vincristine, vinblastine, vindesine, vinzolidine andvinorelbine), camptothecin 20(S)-camptothecin,9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin),podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), andtaxanes (e.g., paclitaxel and docetaxel).

In yet another embodiment, a biologic agent is used in combination withand/or added to the inventive compound/formulation, such asimmuno-modulating proteins such as cytokines, monoclonal antibodiesagainst tumor antigens, tumor suppressor genes, and cancer vaccines.

Examples of interleukins that may be used in combination with and/oradded to the inventive compound/formulation include, but are not limitedto, interleukin 2 (IL-2), and interleukin 4 (IL-4), interleukin 12(IL-12). Examples of interferons that may be used in conjunction withdecitabine-glycerin formulations include, but are not limited to,interferon α, interferon β (fibroblast interferon) and interferon γ(fibroblast interferon). Examples of such cytokines include, but are notlimited to erythropoietin (epoietin), granulocyte-CSF (filgrastim), andgranulocyte, macrophage-CSF (sargramostim). Immuno-modulating agentsother than cytokines include, but are not limited to bacillusCalmette-Guerin, levamisole, and octreotide.

Example of monoclonal antibodies against tumor antigens that can be usedin conjunction with the inventive formulations include, but are notlimited to, HERCEPTIN® (Trastruzumab), RITUXAN® (Rituximab), MYLOTARG®(anti-CD33 antibody), and CAMPATH® (anti-CD52 antibody).

In yet another embodiment, a kinase inhibitor is used in combinationwith and/or added to the inventive compound/formulation for treatingdiseases associated with abnormal kinase activity.

In one variation, the tyrosine kinase inhibitor is imatanib mesylate(e.g., Gleevec®). Imatanib mesylate is a protein tyrosine kinaseinhibitor that inhibits the Bcr-Abl tyrosine kinase created by thePhiladelphia chromosome abnormality in CML. Imatanib mesylate achievesthis inhibitory result through binding to the adenosinetriphosphate-binding site of the Bcr-Abl tyrosine kinase, which preventsphosphorylation of substrates and related malignant transformation.Through inhibition of this kinase, it is believed that imatinib mesylateinhibits cell proliferation and induces apoptosis. T. Schindler et al(2000) Science 289:1938-1942.

In another variation, the kinase is a serine/threonine kinase such as aRaf kinase; and the kinase inhibitor is BAY 43-9006.

In yet another variation, the kinase is a protein kinase such as aRaf-mitogen-activated protein kinase (MEK) and protein kinase B (Akt)kinase.

In yet another variation, the kinase is an extracellularsignal-regulated kinase (ERK). Examples of the inhibitor of ERK includebut are not limited to PD98059, PD184352, and U0126.

In yet another variation, the kinase is a phosphatidylinositol 3′-kinase(PI3K). Examples of the inhibitor of PI3K include but are not limited toLY294002.

In a particular variation, the kinase is a tyrosine kinase. The tyrosinekinase may be a receptor tyrosine kinase and non-receptor tyrosinekinase.

Examples of the receptor tyrosine kinase include, but are not limitedto, epidermal growth factor receptor family (EGFR), platelet-derivedgrowth factor receptor (PDGFR) family, vascular endothelial growthfactor receptor (VEGFR) family, nerve growth factor receptor (NGFR)family, fibroblast growth factor receptor family (FGFR) insulin receptorfamily, ephrin receptor family, Met family, and Ror family.

Examples of the epidermal growth factor receptor family include, but arenot limited to, HER1, HER2/neu, HER3, and HER4.

Examples of the inhibitors of epidermal growth factor receptor familyinclude, but are not limited to, HERCEPTIN®, ZD1839 (IRESSA®), PD168393,CI1033, IMC-C225, EKB-569, and inhibitors binding covalently to Cysresidues of the receptor tyrosine kinase.

Examples of diseases associated with abnormal activity of the epidermalgrowth factor receptor family, include, but are not limited to,epithelial tumor, carcinoma, carcinoma of upper aerodigestive tract,lung cancer, and non-small cell lung cancer.

Examples of the vascular endothelial growth factor receptor familyinclude, but are not limited to, VEGFR1, VEGFR2, and VEGFR3.

An example of the inhibitor of the vascular endothelial growth factorreceptor family includes, but is not limited to, SU6668.

Examples of the disease associated with abnormal activity of thevascular endothelial growth factor receptor family include, but are notlimited to, solid and metastasis-prone tumors.

Examples of the nerve growth factor receptor family include, but are notlimited to, trk, trkB and trkC.

Examples of the inhibitors of the nerve growth factor receptor familyinclude, but are not limited to, CEP-701, CEP-751, and indocarbazolecompound.

Examples of the diseases associated with abnormal activity of the nervegrowth factor receptor family include, but are not limited to, prostate,colon, papillary and thyroid cancers, neuromas and osteoblastomas.

Examples of the Met family include, but are not limited to, Met,TPR-Met, Ron, c-Sea, and v-Sea.

Examples of disease associated with activity of the receptor tyrosinekinase from Met family include, but are not limited to, invasivelyin-growing tumor, carcinoma, papillary carcinoma of thyroid gland,colon, carcinoma, renal carcinoma, pancreatic carcinoma, ovariancarcinoma, head and neck squamous carcinoma.

Examples of the non-receptor tyrosine kinase include, but are notlimited to, c-kit family, Src family, Fes family, JAK family, Fakfamily, Btk family, Syk/ZAP-70 family, and Abl family.

Examples of the non-receptor tyrosine kinases from the Src familyinclude, but are not limited to, Src, c-Src, v-Src, Yes, c-Yes, v-Yes,Fyn, Lyn, Lck, Blk, Hck, Fgr, c-Fgr, v-Fgr, p561ck, Tkl, Csk, and Ctk.

Examples of the inhibitors of the non-receptor tyrosine kinase from theSrc family include, but are not limited to, SU101 and CGP 57418B.

Examples of the diseases associated with activity of the non-receptortyrosine kinase from the Src family include, but are not limited to,breast cancer, carcinoma, myeloma, leukemia, and neuroblastoma.

Examples of the non-receptor tyrosine kinases from the Fes familyinclude, but are not limited to, c-fes/fps, v-fps/fes, p94-c-fes-relatedprotein, and Fer.

Examples of the diseases associated with activity of the non-receptortyrosine kinase from the Fes family include, but are not limited to,tumor of mesenchymal origin and tumor of hematopoietic origin.

Examples of the non-receptor tyrosine kinases from the JAK familyinclude, but are not limited to, Jak1, Jak2, Tyk2, and Jak3.

Examples of the inhibitors of the non-receptor tyrosine kinase from theJAK family include, but are not limited to, tyrphostin, member ofCIS/SOCS/Jab family, synthetic component AG490, dimethoxyquinazolinecompound, 4-(phenyl)-amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline,4-(3′-bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline, and4-(3′,5′-dibromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline.

Examples of the diseases associated with activity of the non-receptortyrosine kinase from JAK family include, but are not limited to, tumorof mesenchymal origin and tumor of hematopoietic origin.

Examples of the non-receptor tyrosine kinases from the Fak familyinclude, but are not limited to, Fak and CAK.beta./Pyk2/RAFTK.

Examples of the inhibitors of the non-receptor tyrosine kinases from theFak family include, but are not limited to, a dominant negative mutantS1034-FRNK; a metabolite FTY720 from Isaria sinclarii, and FAK antisenseoligonucleotide ISIS15421.

Examples of the diseases associated with abnormal activity of thenon-receptor tyrosine kinases from Fak family include, but are notlimited to, human carcinoma, metastasis-prone tumor, and tumor ofhematopoietic origin.

Examples of the non-receptor tyrosine kinase from the Btk familyinclude, but are not limited to, Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Itk,Tec, Bmx, and Rlk.

Examples of the inhibitors of the non-receptor tyrosine kinases from Btkfamily include, but are not limited to,alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide.

Examples of the diseases associated with abnormal activity of thenon-receptor tyrosine kinase from the Btk family include, but are notlimited to, B-lineage leukemia and lymphoma.

Examples of the non-receptor tyrosine kinases from the Syk/ZAP-70 familyinclude, but are not limited to, Syk and ZAP-70.

Examples of the inhibitors of the non-receptor tyrosine kinases from theSyk/ZAP-70 family include, but are not limited to, piceatannol,3,4-dimethyl-10-(3-aminopropyl)-9-acridone oxalate, acridone-relatedcompound, Lys-Leu-Ile-Leu-Phe-Leu-Leu-Leu [SEQ ID NO: 1) peptide, andpeptide containing Lys-Leu-Ile-Leu-Phe-Leu-Leu-Leu motif.

Examples of the diseases associated with abnormal activity of thenon-receptor tyrosine kinases from the Syk/ZAP-70 family include, butare not limited to, benign breast cancer, breast cancer, and tumor ofmesenchymal origin.

5. Indications for Compounds or Pharmaceutical Compositions of thePresent Invention

The pharmaceutical formulations according to the present invention maybe used to treat a wide variety of diseases, preferably those associatedwith aberrant DNA methylation.

Preferable indications that may be treated using the pharmaceuticalformulations of the present invention include those often involvingundesirable or uncontrolled cell proliferation. Such indications includebenign tumors, various types of cancers such as primary tumors and tumormetastasis, restenosis (e.g., coronary, carotid, and cerebral lesions),hematological disorders, abnormal stimulation of endothelial cells(atherosclerosis), insults to body tissue due to surgery, abnormal woundhealing, abnormal angiogenesis, diseases that produce fibrosis oftissue, repetitive motion disorders, disorders of tissues that are nothighly vascularized, and proliferative responses associated with organtransplants.

Generally, cells in a benign tumor retain their differentiated featuresand do not divide in a completely uncontrolled manner. A benign tumor isusually localized and nonmetastatic. Specific types benign tumors thatcan be treated using the present invention include hemangiomas,hepatocellular adenoma, cavernous haemangioma, focal nodularhyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bileduct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas and pyogenicgranulomas.

In a malignant tumor cells become undifferentiated, do not respond tothe body's growth control signals, and multiply in an uncontrolledmanner. The malignant tumor is invasive and capable of spreading todistant sites (metastasizing). Malignant tumors are generally dividedinto two categories: primary and secondary. Primary tumors arisedirectly from the tissue in which they are found. A secondary tumor, ormetastasis, is a tumor which is originated elsewhere in the body but hasnow spread to a distant organ. The common routes for metastasis aredirect growth into adjacent structures, spread through the vascular orlymphatic systems, and tracking along tissue planes and body spaces(peritoneal fluid, cerebrospinal fluid, etc.)

Specific types of cancer or malignant tumors, either primary orsecondary, that can be treated using this invention include breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gall bladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet celltumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullarycarcinoma, pheochromocytoma, mucosal neuronms, intestinalganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

Hematologic disorders include abnormal growth of blood cells which canlead to dysplastic changes in blood cells and hematologic malignanciessuch as various leukemias. Examples of hematologic disorders include butare not limited to acute myeloid leukemia, acute promyelocytic leukemia,acute lymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes, and sickle cell anemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchromosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subgroup ofAML. This subtype is characterized by promyelocytic blasts containingthe 15; 17 chromosomal translocation. This translocation leads to thegeneration of the fusion transcript comprised of the retinoic acidreceptor and a sequence PML.

Acute lymphoblastic leukemia (ALL) is a heterogenerous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality is the 9; 22 translocation. The resultantPhiladelphia chromosome represents poor prognosis of the patient.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferativedisorder of a pluripotent stem cell. CML is characterized by a specificchromosomal abnormality involving the translocation of chromosomes 9 and22, creating the Philadelphia chromosome. Ionizing radiation isassociated with the development of CML.

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia.

Treatment of abnormal cell proliferation due to insults to body tissueduring surgery may be possible for a variety of surgical procedures,including joint surgery, bowel surgery, and cheloid scarring. Diseasesthat produce fibrotic tissue include emphysema. Repetitive motiondisorders that may be treated using the present invention include carpaltunnel syndrome. An example of cell proliferative disorders that may betreated using the invention is a bone tumor.

The proliferative responses associated with organ transplantation thatmay be treated using this invention include those proliferativeresponses contributing to potential organ rejections or associatedcomplications. Specifically, these proliferative responses may occurduring transplantation of the heart, lung, liver, kidney, and other bodyorgans or organ systems.

Abnormal angiogenesis that may also be treated using this inventioninclude those abnormal angiogenesis accompanying rheumatoid arthritis,ischemic-reperfusion related brain edema and injury, cortical ischemia,ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome),endometriosis, psoriasis, diabetic retinopaphy, and other ocularangiogenic diseases such as retinopathy of prematurity (retrolentalfibroplastic), muscular degeneration, corneal graft rejection,neuroscular glaucoma and Oster Webber syndrome.

Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanism ofvarious angiogenic factors, including elements of the inflammatoryresponse, such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

In another embodiment, the pharmaceutical formulations of the presentinvention may be used for treating diseases associated with undesired orabnormal angiogenesis. The method comprises administering to a patientsuffering from undesired or abnormal angiogenesis the pharmaceuticalformulations of the present invention alone, or in combination withanti-neoplastic agent whose activity as an anti-neoplastic agent in vivois adversely affected by high levels of DNA methylation. The particulardosage of these agents required to inhibit angiogenesis and/orangiogenic diseases may depend on the severity of the condition, theroute of administration, and related factors that can be decided by theattending physician. Generally, accepted and effective daily doses arethe amount sufficient to effectively inhibit angiogenesis and/orangiogenic diseases.

According to this embodiment, the pharmaceutical formulations of thepresent invention may be used to treat a variety of diseases associatedwith undesirable angiogenesis such as retinal/choroidalneuvascularization and corneal neovascularization. Examples ofretinal/choroidal neuvascularization include, but are not limited to,Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease,vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis,pseudoxanthoma elasticum carotid abostructive diseases, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, diabeticretinopathy, macular degeneration, Bechets diseases, infections causinga retinitis or chroiditis, presumed ocular histoplasmosis, parsplanitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications, diseases associatedwith rubesis (neovascularization of the angle) and diseases caused bythe abnormal proliferation of fibrovascular or fibrous tissue includingall forms of proliferative vitreoretinopathy. Examples of cornealneuvascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, polyarteritis,Wegener sarcoidosis, Scleritis, periphigoid radial keratotomy,neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections and Kaposi sarcoma.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used for treating chronic inflammatory diseasesassociated with abnormal angiogenesis. The method comprisesadministering to a patient suffering from a chronic inflammatory diseaseassociated with abnormal angiogenesis the pharmaceutical formulations ofthe present invention alone, or in combination with an anti-neoplasticagent whose activity as an anti-neoplastic agent in vivo is adverselyaffected by high levels of DNA methylation. The chronic inflammationdepends on continuous formation of capillary sprouts to maintain aninflux of inflammatory cells. The influx and presence of theinflammatory cells produce granulomas and thus, maintains the chronicinflammatory state. Inhibition of angiogenesis using the pharmaceuticalformulations of the present invention may prevent the formation of thegranulosmas, thereby alleviating the disease. Examples of chronicinflammatory disease include, but are not limited to, inflammatory boweldiseases such as Crohn's disease and ulcerative colitis, psoriasis,sarcoidois, and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis are characterized by chronic inflammation and angiogenesis atvarious sites in the gastrointestinal tract. For example, Crohn'sdisease occurs as a chronic transmural inflammatory disease that mostcommonly affects the distal ileum and colon but may also occur in anypart of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea. Theseinflammatory bowel diseases are generally caused by chronicgranulomatous inflammation throughout the gastrointestinal tract,involving new capillary sprouts surrounded by a cylinder of inflammatorycells. Inhibition of angiogenesis by the pharmaceutical formulations ofthe present invention should inhibit the formation of the sprouts andprevent the formation of granulomas. The inflammatory bowel diseasesalso exhibit extra intestinal manifestations, such as skin lesions. Suchlesions are characterized by inflammation and angiogenesis and can occurat many sites other the gastrointestinal tract. Inhibition ofangiogenesis by the pharmaceutical formulations of the present inventionshould reduce the influx of inflammatory cells and prevent the lesionformation.

Sarcoidois, another chronic inflammatory disease, is characterized as amulti-system granulomatous disorder. The granulomas of this disease canform anywhere in the body and, thus, the symptoms depend on the site ofthe granulomas and whether the disease is active. The granulomas arecreated by the angiogenic capillary sprouts providing a constant supplyof inflammatory cells. By using the pharmaceutical formulations of thepresent invention to inhibit angionesis, such granulomas formation canbe inhibited. Psoriasis, also a chronic and recurrent inflammatorydisease, is characterized by papules and plaques of various sizes.Treatment using the pharmaceutical formulations of the present inventionshould prevent the formation of new blood vessels necessary to maintainthe characteristic lesions and provide the patient relief from thesymptoms.

Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Treatment using thepharmaceutical formulations of the present invention alone or inconjunction with other anti-RA agents may prevent the formation of newblood vessels necessary to maintain the chronic inflammation and providethe RA patient relief from the symptoms.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used for treating diseases associated withabnormal hemoglobin synthesis. The method comprises administering thepharmaceutical formulations of the present invention to a patientsuffering from disease associated with abnormal hemoglobin synthesis.Decitabine containing formulations stimulate fetal hemoglobin synthesisbecause the mechanism of incorporation into DNA is associated with DNAhypomethylation. Examples of diseases associated with abnormalhemoglobin synthesis include, but are not limited to, sickle cell anemiaand β-thalassemia.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used to control intracellular gene expression.The method comprises administering the pharmaceutical formulations ofthe present invention to a patient suffering from disease associatedwith abnormal levels of gene expression. DNA methylation is associatedwith the control of gene expression. Specifically, methylation in ornear promoters inhibit transcription while demethylation restoresexpression. Examples of the possible applications of the describedmechanisms include, but are not limited to, therapeutically modulatedgrowth inhibition, induction of apoptosis, and cell differentiation.

Gene activation facilitated by the pharmaceutical formulations of thepresent invention may induce differentiation of cells for therapeuticpurposes. Cellular differentiation is induced through the mechanism ofhypomethylation. Examples of morphological and functionaldifferentiation include, but are not limited to differentiation towardsformation of muscle cells, myotubes, cells of erythroid and lymphoidlineages.

Although exemplary embodiments of the present invention have beendescribed and depicted, it will be apparent to the artisan of ordinaryskill that a number of changes, modifications, or alterations to theinvention as described herein may be made, none of which depart from thespirit of the present invention. All such changes, modifications, andalterations should therefore be seen as within the scope of the presentinvention.

EXAMPLES

The following examples are representative of the invention, and providedetailed methods for preparing the compounds of the invention. In theseexamples, elemental analyses were carried out in the MicrochemicalLaboratory, University of Otago, Dunedin, NZ. Melting points weredetermined on an Electrothermal 2300 Melting Point Apparatus. NMRspectra were obtained on a Bruker Avance-400 spectrometer at 400 MHz for¹H and 100 MHz for 13C spectra, referenced to Me4Si. Mass spectra weredetermined on a VG-70SE mass spectrometer using an ionizing potential of70 eV at a nominal resolution of 1000. High-resolution spectra wereobtained at nominal resolutions of 3000, 5000, or 10000 as appropriate.All spectra were obtained as electron impact (EI) using PFK as thereference unless otherwise stated. Column chromatography was carried outon silica gel, (Merck 230-400 mesh) unless otherwise stated.

Example A Preparation of1-methyl-4-(3-{[4-(4-quinolinylamino)benzoyl]amino}anilino)pyridiniumchloride (Cpd. A)

N-(3-Nitrophenyl)-4-pyridinamine (A3). A suspension of p-toluenesulfonicacid monohydrate (17.4 g, 0.126 mol) in benzene was azeotroped for 10 h.Phenol (50 g) was added and the mixture was azeotroped for 2 h, then4-pyridylpyridinium chloride (26.6 g, 0.138 mmol) and 3-nitroaniline(17.4 g, 0.126 mmol) was added and the mixture was azeotroped for 3 h.Benzene was removed under reduced pressure and the resulting blackresidue was heated to 180° C. for 1 h. The reaction mixture was cooledto 20° C., and 4N NaOH (150 mL) was added. The mixture was stirred for30 min, and then diluted with water (2 L). This mixture was stirred inCH₂Cl₂ (1 L) and the resulting precipitate was filtered and washed withmore CH₂Cl₂. The solid was recrystallized from MeOH/H₂O to give A3 (11.9g), as a yellow solid: mp (MeOH/H₂O) 182-184° C. Further material wasrecovered from CH₂Cl₂ washes, by drying over Na₂SO₄ and evaporation todryness. The residue was dissolved in CH₂Cl₂ (100 mL) and hexanes (200mL) were added, and the mixture was stirred for 16 h. The resultingprecipitate was filtered and washed with ether to remove any unreacted3-nitroaniline, and the solid was crystallized from MeOH/H₂O to giveadditional A3 (3.2 g; total yield 15.7 g, 58%); ¹H NMR [(CD₃)₂SO] δ 9.28(s, 1H, NH), 8.31-8.29 (m, 2H, H-2, 6), 7.95 (t, J=2.1 Hz, 1H, H-2′),7.82-7.79 (m, 1H, H-4′), 7.65-7.57 (m, 2H, H-5′, H-6′), 7.03-7.02 (m,2H, H-3, H-5), ¹³C NMR [(CD₃)₂SO] δ 150.5 (2×C), 148.8, 148.6, 142.2,130.8, 124.8, 116.2, 112.7, 110.2 (2×C); Analysis calc. for C₁₁H₉N₃O₂:C, 61.4; H, 4.2; N, 19.5; found, C, 64.5; H, 4.3; N, 19.7%.

N¹-(4-Pyridinyl)-1,3-benzenediamine (A4). A suspension of compound A3(7.26 g, 33.7 mmol) and 10% Pd/C in MeOH was hydrogenated for 2 h,filtered through a pad of Celite, and solvent evaporated. The residuewas recrystallized from MeOH/H₂O to give A4 as a pale yellow powder(5.43 g, 83%): mp. (MeOH/H₂O) 170-171° C. ¹H NMR [(CD₃)₂SO] δ 8.48 (bs,1H, NH), 8.14-8.12 (m, 2H, H-2′&6′), 6.96 (t, J=7.9 Hz, 1H, H-5),6.86-6.84 (m, 2H, H-3′ & 6′), 6.43 (t, J=2.0 Hz, 1H, H-2), 6.32 (dd,J=7.8, 1.3H, 1H, H-6), 6.26 (dd, J=7.8, 1.5 Hz, 1H, H-4) 5.07 (br s, 2H,NH₂); HRMS (EI⁺) calc. for C₁₁H₁₁N₃ (M⁺) m/z 185.0953, found 185.0945;Anal. calc. for C₁₁H₁₁N₃.0.125H₂O: C, 70.5; H, 6.1; N, 22.5; found, C,70.6; H, 6.0; N, 22.7%.

N-(4-Nitrophenyl)-3-(4-pyridinylamino)benzamide (A5). 4-Nitrobenzoicacid (4.3 g, 16.36 mmol) was suspended in SOCl₂ (30 mL), 2 drops of DMFwere added, and the mixture was refluxed for 1 h (until a clear solutionwas obtained). The reaction mixture was cooled to room temperature andexcess SOCl₂ was removed under vacuum. The resulting residue wasdissolved in 1,4-dioxane and added to a suspension of A4 (3.0 g, 16.20mmol) in 1,4-dioxane (300 mL) containing pyridine (8 mL). The reactionmixture was stirred 16 h at 50° C., and the solvent was then evaporated.The residue was stirred in dilute aqueous ammonia, and the resultingprecipitate was filtered and crystallized from MeOH to give A5 (5.3 g,79%): mp (MeOH) 219-222° C.; ¹H NMR [(CD₃)₂SO] δ 10.63 (s, 1H, NH), 9.04(s, 1H, NH), 8.37 (d, J=8.9 Hz, 2H, ArH), 8.24-8.18 (m, 4H, ArH), 7.81(bs, 1H, ArH), 7.45 (d, J=8.5 Hz, 1H, ArH), 7.34 (t, J=8.0 Hz, 1H, ArH),6.99-6.95 (m, 3H, ArH), ¹³C NMR [(CD₃)₂SO] δ 166.9, 164.0, 150.3, 149.4(2×C), 149.1, 140.5, 139.6, 129.5 (2×C), 129.2, 123.5 (2×C), 123.4,115.8, 114.6, 111.6, 109; HRMS (FAB⁺) calc. for C₁₈H₁₅N₄O₃ (M⁺¹) m/z335.1144, found 335.1154.

1-Methyl-4-{3-[(4-nitroanilino)carbonyl]anilino}pyridinium chloride(A6). To a solution of A5 (507 mg, 1.51 mmol) in DMF (2 mL) was addedmethyl-p-toluene sulfonate (3 mL), and the reaction mixture was stirredat room temperature for 20 h. Solvent was removed under reducedpressure, and the residue re-dissolved in MeOH. This solution wasevaporated to dryness, and the residue crystallized from MeOH/EtOAc togive A6 as a tosylate salt (530 mg, 67%). This was converted to achloride salt by ion-exchange, as follows. AG^(R)1-X₄ resin 200-400chloride form (7 g) was washed with water and packed in a column.Tosylate A6 (530 mg) was stirred in pre-washed resin (2 g), and theresulting slurry was loaded onto the column. The column was then elutedwith water, and fractions containing the compound were combined andevaporated to dryness. The residue was azeotroped with MeOH (3×20 mL),and finally reprecipitated from MeOH/EtOAc to give A6 as the chloridesalt (317 mg 54%): mp (MeOH/EtOAc) 295-298° C. ¹H NMR [(CD₃)₂SO] δ 10.82(s, 1H, NH), 10.76 (s, 1H, NH), 8.38 (d, J=8.9 Hz, 2H, ArH), 8.31 (d,J=7.5 Hz, 2H, ArH), 8.21 (d, J=8.9 Hz, 2H, ArH), 7.96 (t, J=1.9 Hz, 1 H,ArH), 7.64 (dd, J=8.7, 1.1 Hz, 1H, ArH), 7.50 (t, J=5.4 Hz, 1H, ArH),7.22 (d, J=7.6 Hz, 2H, ArH), 7.11 (dd, J=7.9, 1.3 Hz, 1H, ArH), 3.97 (s,3H, N⁺CH₃); HRMS (FAB⁺) calc. for C₁₉H₁₇N₄O₃ (M⁺¹) m/z 349.1301, found349.1303.

4-{3-[(4-Ammoniobenzoyl)amino]anilino}-1-methylpyridinium dichloride(A7). A6 (1.57 g, 4.08 mmol) was dissolved in 5:1H₂O:EtOH (62 mL), Fedust was added (1.1 g), and the resulting suspension was refluxed withvigorous stirring for 5 h. The hot reaction mixture was filtered througha pad of Celite, and the Celite pad was washed with hot EtOH. Thecombined EtOH fractions were evaporated to dryness, and the residueextracted with warm water. This solution was evaporated to dryness, andthe residue was dried by azeotroping with methanol (3×30 mL). Theresidue was dissolved in MeOH (20 mL), methanolic HCl (1.25 M, 5 mL) wasadded, and the solution was stirred for 30 min. The solution wasevaporated to dryness, and the residue dried by azeotroping with MeOH(3×30 mL). The residue was finally crystallized from MeOH/EtOAc to giveA7 (913 mg, 63%) as a white solid: mp (MeOH/EtOAc) 269-273° C.; ¹H NMR[(CD₃)₂SO] δ 10.76 (s, 1H, NH), 10.05 (s, 1H, NH), 8.29 (d, J=7.4 Hz,2H, ArH), 7.95 (t, J=1.9 Hz, 1H, ArH), 7.78 (d, J=8.7 Hz, 2H, ArH), 7.60(dd, J=8.7, 1.1 Hz, 1H, ArH), 7.60 (t, J=8.1 Hz, 1H ArH), 7.20 (d, J=7.5Hz, 2H, ArH), 7.00 (dd, J=7.9, 1.4 Hz, 1H, ArH), 6.72 (d, J=8.6 Hz, 2H,ArH), 3.96 (s, 3H, N⁺CH₃) the signal for NH₂ was not observed; HRMS(FAB⁺) calc. for C₁₉H₁₉N₄O (M⁺) m/z 319.1559, found 319.1562. AnalysisCRL11720 calc. CHN for C₁₉H₂₀N₄Cl₂O: C, 58.3; H, 5.4; N, 14.3; found, C,58.7; H, 5.3; N, 14.5%.

1-Methyl-4-(3-{[4-(4-quinolinylamino)benzoyl]amino}anilino)-pyridiniumchloride (Cpd. A). To a suspension of A7 (200 mg, 0.51 mmol) in MeOH (20mL) was added 4-chloroquinoline (100 mg, 0.61 mmol), and the mixture washeated at reflux for 1 h (until a clear solution was obtained). A dropof c. HCl was then added, and refluxing was continued for a further 20h. The reaction mixture was evaporated to dryness, and the residue wasdissolved in MeOH (10 mL). EtOAc (50 mL) was then added, and the MeOHwas boiled off. The resulting precipitate was filtered, washed withEtOAc, and crystallized from MeOH/EtOAc to give Cpd. A (214 mg, 81%) asa pale yellow solid: mp 253-257° C.; ¹H NMR [(CD₃)₂SO] δ 11.07 (br, 1H,NH), 10.79 (s, 1H, NH), 10.60 (s, 1H, NH), 8.84 (d, J=13.5 Hz, 1H, ArH),8.61 (d, J=6.9 Hz, 1H, ArH), 8.31 (d, J=7.4 Hz, 2H, ArH), 8.18 (d, J=8.6Hz, 2H, ArH), 8.12-8.04 (m, 2H, ArH), 7.99 (br s, 1H, ArH), 7.84 (t,J=7.2 Hz, 1H, ArH), 7.68 (d, J=8.5 Hz, 2H, ArH), 7.49 (t, J=8.1 Hz, 1H,ArH), 7.23 (d, J=7.5 Hz, 2H, ArH), 7.10 (br d, J=7.8 Hz, 1H, ArH), 7.01(d, J=6.7 Hz, 1H, ArH), 3.98 (s, 3H, N⁺CH₃; ¹³C NMR [(CD₃)₂SO] δ 164.8,154.6, 154.3, 144.2, 142.8, 140.5, 140.3, 138.3, 137.3, 133.8, 132.5,129.8, 129.4 (2×C), 127.0, 124.4 (2×C), 124.1, 120.2, 118.0, 117.9,117.5, 114.5, 109.2, 100.3, 44.6, enhancement of two of the carbonsignals was difficult to observe; HRMS (FAB⁺) calc. For C₂₈H₂₄N₅O446.1981, found 446.1975; Anal. Calc.CHN for C₂₈H₂₅Cl₂N₅O.2.25H₂O: C,60.2; H, 5.3; N, 12.5; found, C, 60.1; H, 5.3; N, 12.5%.

Example B Preparation of1-methyl-4-(4-(4-(quinolin-4-ylamino)benzamido)phenylamino)pyridiniumchloride hydrochloride (Cpd. B)

N-4-Nitrophenyl)-4-pyridinamine (B3). 4-Toluenesulfonic acid (53.80 g,0.28 mol) was dissolved in benzene (200 mL), and the resulting solutionrefluxed under Dean-Stark conditions for approximately 96 h, untilevolution of H₂O ceased. To this solution was added phenol (112.25 g,1.19 mol), and the resulting mixture refluxed under Dean-Starkconditions for approximately 1 h, until evolution of H₂O ceased. Afterthis time, 1-(4-pyridyl)-pyridinium chloride (59.95 g, 0.31 mol) (A2)and 4-nitroaniline (B1) were added, and the resulting mixture wasrefluxed under Dean-Stark conditions for approximately 2 h, untilevolution of H₂O ceased. After this time, benzene was removed underreduced pressure, and the resulting black residue heated to 180° C. for2 h. The residue was then cooled to room temperature, and basifiedthoroughly by addition of 4 N aqueous NaOH. The resulting solution wasdiluted with H₂O and CHCl₂, and stirred for 2 h at room temperature. Theresulting suspension was filtered through a pad of Celite to give afirst batch of nitroaniline B3 (0.22 g) as a fine yellow solid. Thefiltrate was diluted with MeOH, re-basified with 4 N NaOH, and thenextracted with EtOAc (×4). The combined organic extracts were washedwith H₂O (×1), brine, and dried over MgSO₄. Solvent was removed underreduced pressure to afford a second batch of B3 (55 g); ¹H NMR[(CD₃)₂SO]: δ 7.16 (dd, J=4.76, 1.60 Hz, 2H, ArH), 7.33 (ddd, J=10.37,5.32, 3.23 Hz, 2H, ArH), 8.18 (ddd, J=10.37, 5.32, 3.23 Hz, 2H, ArH),8.38 (dd, J=4.72, 1.56 Hz, 2H, ArH), 9.71 (br s, Ar—NH—Ar). LCMS(APCI⁺): 216 (100%).

N¹-(4-Pyridinyl)-1,4-benzenediamine (B4). Nitroaniline B3 (˜55.00 g,˜0.26 mol) was brought to reflux in 2:1 EtOH:H₂O (500 mL), and then Fedust (56.96 g, 1.02 mmol) and glacial acetic acid (10 mL) were added.The resulting mixture was refluxed for 30 min, and then cooled to roomtemperature. The reaction mixture was basified by addition of aqueousNH₃, and filtered to remove solids. Solvent was removed under reducedpressure to give B4 as a fluffy, crystalline magenta solid (28.49 g, 54%from B1); ¹H NMR [(CD₃)₂SO]: δ 4.99 (br s, 2H, Ar—NH₂), 6.58 (m, 4H,ArH), 6.86 (ddd, J=9.66, 4.99, 3.04 Hz, 2H, ArH), 8.03 (d, J=6.28 Hz,2H, ArH), 8.24 (s, 1H, Ar—NH—Ar). LCMS (APCI⁺): 186 (100%).

N-(4-Nitrophenyl)-4-(4-pyridinylamino)benzamide (B5). To a solution ofB4 (10.07 g, 54.37 mmol) in dry pyridine (21.90 mL, 271.82 mmol) wasadded a solution of 4-nitrobenzoyl chloride (10.09 g, 54.37 mmol) in drydioxane (100 mL), and the resulting mixture was heated at 50° C. for 2h. After this time, the reaction mixture was cooled to room temperatureand basified by addition of aqueous NH₃. The resulting precipitate wascollected by filtration to give a first batch of amide B5 (3.47 g, 19%)as an amorphous orange-yellow solid. The filtrate was extracted withEtOAc (×4), and the combined organic extracts were washed with brine,and then evaporated to dryness to give a mixture of B4 and 135 (ca. 1:1by ¹H NMR). Re-treatment of this mixture with 4-nitrobenzoyl chloride at50° C. for 12 h gave a further 4 g of B5; ¹H NMR [(CD₃)₂SO]: δ 6.86 (dd,J=4.82, 1.60 Hz, 2H, ArH), 7.21 (dt, J=9.86, 5.02, 3.00 Hz, 2H, ArH),7.76 (d, J=8.84, 2H, ArH), 8.18 (m, 4H, ArH), 8.37 (ddd, J=9.23, 4.33,2.34 Hz, 2H, ArH), 8.73 (s, 1H, ArNHAr), 10.52 (s, 1H, ArC(O)NHAr). LCMS(APCI⁺): 335 (100%).

1-Methyl-4-{4-[(4-nitroanilino)carbonyl]anilino}pyridinium chloride(B6). o a solution of amide B5 (5.62 g, 16.82 mmol) in dry DMF (110 mL)was added methyl tosylate (33.00 mL, 218.68 mmol), and the resultingmixture was stirred at room temperature for 12 h. After this time, thereaction mixture was filtered through a pad of Celite to give a firstbatch of the tosylate salt of 11 as an amorphous bright yellow solid(7.57 g, 86%). The filtrate was concentrated under reduced pressureuntil a precipitate formed, and this was collected by filtration througha pad of Celite to give a second batch of the tosylate salt of B6 (0.72g, 8%). ¹H NMR [(CD₃)₂SO]: δ 2.29 (s, 3H, ⁻O(O)₂SPhCH₃), 3.96 (s, 3H,R₂N⁺⁻CH₃), 7.10 (m, 4H, O(O)₂SArHCH₃), 7.35 (d, J=8.84 Hz, 2H, ArH),7.47 (d, J=8.08 Hz, 2H, ArH), 7.91 (d, J=8.86, 2H, ArH), 8.20 (dd,J=6.94, 1.94, ArH), 8.26 (d, J=7.45 Hz, 2H, ArH), 8.39 (m, 2H, ArH),10.42 (s, 1H, ArNHAr), 10.70 (s, 1H, ArNHAr). (APCI⁺): 349 (100%).

To a suspension of the tosylate salt of B6 (8.30 g, 15.97 mmol) in MeOH(˜200 mL), was added ion-exchange resin (67 g, pre-washed with H₂O)[1-X₄, BioRad AG, 200-400 chloride form]. The resulting suspension wasloaded onto a column of more ion-exchange resin (70 g, pre-washed withH₂O), and the column was eluted with MeOH. Solvent was removed underreduced pressure to afford chloride salt of B6 (6.47 g, 91%) as anamorphous yellow solid; ¹H NMR [(CD₃)₂SO]: δ 3.95 (s, 3H, R₂N⁺⁻CH₃),7.10 (m, 2H, ArH), 7.35 (d, J=7.93 Hz, 2H, ArH), 7.91 (d, J=8.52 Hz, 2H,ArH), 8.22 (m, 4H, ArH), 8.39 (d, J=8.76 Hz, 2H, ArH), 10.50 (br s, 1H,ArNHAr), 10.72 (s, 1H, ArNHAr). LCMS (APCI⁺): 349 (100%).

4-{4-[(4-Ammoniobenzoyl)amino]anilino}-1-methylpyridinium dichloride(B7). To a refluxing suspension of amide B6 (6.47 g, 16.81 mmol) in 2:1EtOH:H₂O (600 mL) were sequentially added Fe dust (3.76 g, 67.24 mmol)and glacial acetic acid (12 mL). The resulting mixture was refluxed for2 h, and the hot reaction mixture was then filtered through a pad ofCelite. Solvent was removed under reduced pressure, and the residue wasre-dissolved in MeOH, and solvent again removed under reduced pressure.This latter process was repeated twice more, with a few drops ofmethanolic HCl (˜4M) added in the final re-dissolution. Solvent wasremoved under reduced pressure, and the residue was recrystallized fromMeOH:EtOAc to give amine B8 (6.35 g) as a khaki green-white solid, whichwas used without purification. ¹H NMR [(CD₃)₂SO]: δ 3.94 (s, 3H,R₂N⁺⁻CH₃), 6.85 (d, J=8.26 Hz, 2H, ArH), 7.12 (d, J=7.12 Hz, 2 h, ArH),7.29 (m, 2H, ArH), 7.83 (d, J=8.64 Hz, 2H, ArH), 7.89 (m, 2H, ArH), 8.24(d, J=7.42 Hz, 2H, ArH), 10.10 (s, 1H, ArNHAr, 10.73 (s, 1H, ArH). LCMS(APCI⁺): 319 (100%).

1-Methyl-4-[4-({4-[(6-nitro-4-quinolinyl)amino]benzoyl}amino)anilino]pyridiniumchloride (Cpd. B). 4-Chloroquinoline (89 mg, 0.55 mmol) and 3 drops ofcHCl were sequentially added to a solution of amine B7 (214 mg, 0.55mmol) in dry MeOH (19 mL), and the resulting mixture was then refluxedfor 31 h. After this time, solvent was removed under reduced pressure,and the resulting residue dried via two MeOH-azeotrope cycles. Theresidue was crystallized twice from MeOH:EtOAc, and then furtherpurified by preparative HPLC, to afford Cpd. B as an amorphous yellowsolid (68 mg, 24%). ¹H NMR [(CD₃)₂SO]: 3.96 [s, 3H, ArN⁺CH₃], 7.00 [d,J=6.92 Hz, 1H, ArH], 7.14 [d, J=7.29 Hz, 2H, ArH], 7.35 [d, J=8.86 Hz,2H, ArH], 7.69 [d, J=8.58 Hz, 2H, ArH], 7.85 [t, J=15.42, 7.71 Hz, 1H,ArH], 7.96 [d, J=8.86, 2H, ArH], 8.07 [t, J=15.42, 7.71 Hz, 1H, ArH],8.13 [d, J=7.76 Hz, 1H, ArH], 8.20 [d, J=8.58 Hz, 2H, ArH], 8.26 [d,J=7.39 Hz, 2H, ArH], 8.61 [d, J=6.92 Hz, 1H, ArH], 8.88 [d, J=8.43 Hz,1H, ArH], 10.58 [s, 1H, ArNHAr], 10.76 [s, 1H, ArNHAr], 11.16 [s, H1,ArC(O)NHAr], 14.81 [br s, quinoline-N⁺H]. LCMS (APCI⁺): 447 (100%).HPLC: 99.7%.

Example C Preparation of4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]quinoliniumdichloride (Cpd. C)

(Acetylamino)-N-(4-nitrophenyl)benzamide (C2). A mixture of3-acetamidobenzoic acid (C1) (5.30 g, 29.58 mmol) and CD1 (5.75 g, 35.50mmol, 1.2 equivalent) in N-methylpyrrolidinone (20 mL) was heated at55-60° C. for 2 h. 4-Nitroaniline (6.13 g, 44.37 mmol, 1.5 equivalent)was then added and the mixture was heated to 140° C. for 4 h, thenpoured into water (400 mL) and stirred for 18 h. The resultingprecipitate was filtered and washed sequentially with water and CH₂Cl₂and then crystallized from MeOH to give C2 (4.84 g; 55%) as a yellowsolid; mp (MeOH) 259-261° C.; ¹H NMR [(CD₃)₂SO] δ 10.80 (s, 1H, NH),10.15 (s, 1H, NH), 8.29 (m, 2H, H-{acute over (3)} & H{acute over (5)}),8.12 (t, J=1.8 Hz, 1H, H-2), 7.84 (br dd, J=8.1, 1.2 Hz, 1H, H-6), 7.65(td, J=7.8, 2.5 Hz, 1H, H-4), 7.48 (t, J=7.9 Hz, 1H, H-5), 2.08 (s, 3H,COCH₃); Anal. Calc. For C₁₅H₁₃N₃O₄: C, 60.2; H, 4.4; N, 14.0; found C,60.4; H, 4.6; N, 14.1%.

3-Amino-N-(4-nitrophenyl)benzamide (C3). Compound C2 (4.25 g, 15.9 mmol)was suspended in 1,4-dioxane (100 mL), dilute HCl (15 mL c.HCl+85 mLH₂O) was added, and the mixture was refluxed for 6 h (until TLC in 6%MeOH:CH₂Cl₂ showed complete consumption of starting material). Thereaction mixture was then cooled to 20° C. and the solvent wasevaporated to dryness. The resulting residue was stirred in diluteaqueous NH₃, then filtered, washed with water, oven-dried andcrystallized from MeOH to give amine C3 (2.75 g, 67%); mp (MeOH)229-232° C.; ¹H NMR [(CD₃)₂SO] δ 10.63 (s, 1H, CONH), 8.27-8.22 (m, 2H,H-3′& H-5′), 8.06-8.02 (m, 2H, H-2′ & H-6′), 7.18 (t, J=7.7 Hz, 1H,H-5), 7.12-7.08 (m, 2H, H-2&H-6), 6.80-6.78 (m, 1H, H-4), 5.38 (s, 2H,NH₂); HRMS (FAB⁺) calc. for C₁₃H₁₁N₃O₃ (M⁺¹) m/z 258.0879, found258.0875; Anal. Calc. for C₁₃H₁₁N₃O₃: C, 60.7; H, 4.3; N, 16.3; found C,60.7; H, 4.4; N, 16.6%.

N-(4-Nitrophenyl)-3-(4-pyridinylamino)benzamide (C4).Para-toluenesulfonic acid (2 g, 10.49 mmol) was azeotroped with benzene(250 mL) for 2 h. Pyridylpyridinium chloride (3.3 g), compound C3 (2.7g, 10.49 mmol) and N-methylpyrrolidinone (10 mL) were then added, andthe mixture azeotroped at 130° C. for 2 h. Solvent was boiled off, andthe resulting dark brown mixture was heated at 180° C. for 1 h, and thencooled about 50° C. and diluted with water. The mixture was stirred atroom temperature for 2 h, and the resulting precipitate was filtered andwashed sequentially with water, dilute aqueous NH₃ and CH₂Cl₂. The solidwas then crystallized from MeOH, filtered, and washed with MeOH andCH₂Cl₂ to give C4 (1.92 g, 55%); ¹H NMR [(CD₃)₂SO] δ 10.79 (s, 1H,CONH), 9.00 (s, 1 H, NH), 8.29-8.22 (m, 4H, H-3′, 5′& py H-2, 6)),8.08-8.04 (m, 2H, H-2′, 6′), 7.76 (t, J=1.8 Hz, 1H, H-2), 7.63 (t d,J=8.0, 1.3H, Hz, 1H, H-6), 7.52 (t, J=7.8 Hz, 1H, H-5), 7.46-7.43 (m,1H, H-4), 6.98-6.97 (m, 2H, py H-3&5).

1-Methyl-4-{3-[(4-nitroanilino)carbonyl]anilino}pyridinium4-methylbenzenesulfonate (C5). Compound C4 (1.92 g, 5.73 mmol) wassuspended in DMF (10 mL), methyl-p-toluenesulfonate (15 mL) was added,and the mixture was stirred at room temperature for 18 h. Solvent wasremoved under reduced pressure and the residue was dissolved in warmMeOH (10 mL), then diluted with EtOAc. Some of the MeOH was boiled off,and the solution was then refrigerated for 18 h. The resultingprecipitate was filtered, and washed with EtOAc to give essentially pureCS (2.90 g, 97%), which was used without further purification. ¹H NMR[(CD₃)₂SO] δ 10.85 (s, 1H, CONH), 10.60 (s, 1 H, NH), 8.33-8.27 (m, 4H,H-3′, 5′ & py H-2, 6), 8.08-8.30 (m, 2H, H-2′, 6′), 7.94-7.89 (m, 2H,H-2″ 4″), 7.69 (t, J=7.9 Hz, 1H, H-5″), 7.60-7.57 (m, 1H, H-6″),7.48-7.45 (m, 2H, H-3, 5), 7.22 (d, J=7.5 Hz, 2H, p-tol H-2, 6), 7.20(d, J=8.1 Hz, 2H, p-tol H-3, 5), 3.99 (s, 3H, N⁺CH₃), 1.99 (s, 3H, CH₃).

4-{3-[(4-aminoanilino)carbonyl]anilino}-1-methylpyridinium chloride(C6). Compound CS (1.59 g, 3.06 mmol) was dissolved in ˜6:1 EtOH:H₂O (46mL), Fe dust (885 mg) was added, and the resulting suspension brought toreflux. Two drops of c.HCl were added, and refluxing was continued for 1h. (until TLC with the top phase of 5:4:1 mixture of n-BuOH:H₂O:CH₃CO₂Hshowed complete consumption of starting material). The reaction mixturewas diluted with EtOH (100 mL) and brought to reflux. The hot reactionmixture was filtered through a pad of Celite, and the top layer of theCelite pad was boiled with more EtOH and filtered (this procedure wasrepeated three times to ensure complete extraction of product from ironresidues). The combined EtOH extracts were evaporated to dryness, andthe residue was extracted with hot water, and filtered through a pad ofCelite. The filtrate was concentrated to a smaller volume, and thenstirred in pre-washed ion-exchange resin (55 g). The resulting slurrywas loaded onto a column packed with pre-washed ion-exchange resin andeluted with water. Fractions containing product were combined andevaporated to dryness, and the residue was azeotroped several times withEtOH. The residue was dissolved in a small volume of MeOH, methanolicHCl (1.25 M, 1 mL) was added, and the solution stirred for 10 min. Thesolution was diluted with EtOAc, and the resulting precipitate filteredand then crystallized from MeOH/EtOAc to give C6 (873 mg, 80%); mp(MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 10.77 (br s, 1H, NH), 9.95 (s,1H, NH), 8.41 (br d, J=5.5 Hz, 2H, Py H-2, 6), 7.87-7.84 (m, 2H, H-2,4), 7.61 (t, J=7.8 Hz, 1H, H-5), 7.49 (br d, J=7.8 Hz, 1H, H-6), 7.37(d, J=8.7 Hz, 2H, H-2′, 6′), 7.21 (br d, J=4.6 Hz, 2H py H-3, 5), 6.55(d, J=8.8 Hz, 2H, H-3′, 5′), 4.93 (s, 2H, NH₂), 3.97 (s, 3H, N⁺CH₃).

4-[4-({3-[(1-Methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]quinoliniumdichloride (Cpd. C). Compound C6 (100 mg, 0.28 mmol) was dissolved inEtOH (10 mL) and H₂O (5 mL) by heating. 4-Chloroquinoline (60 mg, 0.36mmol) and 3 drops of c. HCl were added, and the mixture refluxed for 18h (until TLC with the top phase of 5:4:1 mixture of n-BuOH:H₂O:CH₃CO₂Hshowed complete consumption of starting amine). The reaction mixture wasthen diluted with EtOAc, boiled for a few minutes and then allowed tocool to room temperature. The resulting precipitate was filtered andcrystallized from MeOH/EtOAc to give Cpd. C (64 mg, 63%); mp (MeOH,EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 14.49 (br, 1H, N⁺H), 11.01 (s, 1H,NH), 11.78 (br, 1H, NH), 10.64 (s, 1H, NH), 8.77 (d, J=8.5 Hz, 1H, ArH),8.57 (d, J=6.5 Hz, 1H, ArH), 8.32 (d, J=7.5 Hz, 2H, ArH), 8.06-7.92 (m,6H, ArH), 7.53 (t, J=7.7 Hz, 1H, ArH), 7.70-7.65 (m, 1H, ArH), 7.48 (d,J=8.8 Hz, 2H, ArH), 7.29 (d, J=7.5 Hz, 2H, ArH), 6.79 (d, J=6.8 Hz, 1H,ArH), 3.98 (s, 3H, N⁺CH₃).

Example D Preparation of4-[4-({4-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]quinoliniumdichloride (Cpd. D)

4-(4-Pyridinylamino)benzoic acid (D2). Para-toluenesulfonic acid (17.4g, 0.126 mol) was azeotroped with benzene (300 mL) for 5 h.4-Pyridylpyridinium chloride (26.6 g, 0.138 mol), 4-aminobenzoic acid(DO (17.3 g, 0.126 mol) and N-methylpyrrolidinone (40 mL) were thenadded, and the mixture was azeotroped at 110° C. for 1 h in an oil bath.Benzene was evaporated at 130° C., and the temperature was thenincreased to 180° C. for 1.5 h. The reaction mixture was then cooled toroom temperature and diluted with brine. The resulting mixture waswarmed to obtain a precipitate, which was filtered and then oven-driedat 110° C. The solid was then extracted into boiling ethanol (5×150 mL),and the combined EtOH extracts were evaporated to dryness. The resultingresidue was stirred in dilute aqueous NH₃, and the resulting precipitatewas filtered. Further product was obtained by neutralizing the filtratewith glacial AcOH, and filtering the resulting precipitate. Crudeproduct batches were combined and crystallized from H₂O to give D2 (8.8g, 32%): mp (H₂O) 333-336° C.; ¹H NMR [(CD3)2SO] δ 12.05 (br., 1H,COOH), 9.17 (s, 1H, NH), 8.28-8.27 (m, 2H, ArH), 7.90-7.86 (m, 2 H,ArH), 7.26-7.23 (m, 2H, ArH), 7.05-7.03 (m, 2H, ArH), ¹³C NMR [(CD3SO)₂]δ 166.8, 150.2 (2×C), 148.6, 145.0, 130.9 (2×C), 123.4, 117.32 ((2×C),110.6 (2×C); LCMS 215+ve.

N-(4-nitrophenyl)-4-(4-pyridinylamino)benzamide (D3). Compound D2 (172mg, 0.80 mmol) was refluxed in SOCl₂ (5 mL) containing a catalyticamount of DMF for 1 h (until a clear solution was obtained). Thereaction mixture was cooled to room temperature and excess SOCl₂ wasremoved under vacuum. Dioxane (10 mL) was added to the residue, and thenremoved under vacuum. The residue was cooled in a dry ice-acetone bath,p-nitroaniline (112 mg, 0.81 mmol) and pyridine (1.3 mL) were added,followed by Et₃N (0.3 mL), and the mixture was stirred at roomtemperature for 20 min, and then refluxed for 1 h. The reaction mixturewas cooled to room temperature and diluted with H₂O, and then basifiedwith aqueous NH₃ and stirred for 30 min. The resulting precipitate wasfiltered, and washed sequentially with water, hexane and CH₂Cl₂. Thecrude product was dissolved in MeOH and then adsorbed onto silica gel,and the resulting adsorbate chromatographed on silica gel, eluting with0-10% MeOH:CH₂Cl₂, to give D3 (85 mg, 32%); mp 298-302° C. (MeOH); ¹HNMR [(CD₃)₂SO] δ 10.64 (s, 1H, NH), 9.21 (s, 1H, NH), 8.30-8.24 (m, 4H,ArH), 8.09-8.05 (m, 2H, ArH), 8.00-7.98 (m, 2H, ArH), 7.34-7.31 (m, 2H,ArH), 7.07-7.05 (d, m, 2H, ArH); LCMS 235+ve.

1-Methyl-4-{4-[(4-nitroanilino)carbonyl]anilino}pyridinium chloride(D4). Compound D3 (80 mg, 0.24 mmol) was dissolved in DMF (0.8 mL),methyl-p-toluenesulphonate (0.5 mL) was added, and the mixture wasstirred at room temperature for 18 h. Solvent was evaporated underreduced pressure, and the residue was dissolved in MeOH (1 mL), and thendiluted with EtOAc (50 mL). The precipitate which formed upon coolingwas filtered and then crystallized from MeOH/EtOAc to give D5 (119 mg)as the tosylate salt. This was converted to the chloride salt byion-exchange, as follows. AG^(R)1-X₄ resin 200-400 chloride form (3 g)was washed with water and packed in a column. Tosylate D5 (119 mg) wasstirred in pre-washed resin (1 g), and the resulting slurry was loadedonto the column. The column was then eluted with 50% MeOH:H₂O, andfractions containing the compound were combined and evaporated todryness. The residue was azeotroped with MeOH (3×20 mL), and finallyreprecipitated from MeOH/EtOAc to give D4 as the chloride salt (82 mg,88%); mp (MeOH/EtOAc); ¹H NMR [(CD₃)₂SO] δ 11.00 (s, 1H, NH), 10.87 (s,1H, NH), 8.37 (d, J=7.3 Hz, 2H ArH), 8.30-8.26 (m, 2H, ArH), 8.15-8.08(m, 4H, ArH), 7.52 (d, J=8.6 Hz, 2H, ArH), 7.33 (d, J=7.2 Hz, 2H, ArH),4.01 (s, 3H, N⁺CH₃); LCMS 349+ve.

4-{4-[(4-Aminoanilino)carbonyl]anilino}-1-methylpyridinium chloride(D5). Compound D4 (295 mg, 0.77 mmol) was dissolved in ˜5:1 EtOH:H₂O (12mL), and Fe dust (209 mg) was added. The mixture was vigorously stirredand refluxed for 4 h, and then filtered hot through a pad of Celite. TheCelite was washed with boiling EtOH, and EtOH fractions were combinedand evaporated to dryness. The residue was extracted into hot water, andfiltered through Celite, and then evaporated to dryness. The residue wasazeotroped with MeOH (3×20 mL), and then dissolved in MeOH (10 mL).Methanolic HCl (1.25 M, 5 mL) was added, and the solution stirred for 10min. Solvent was removed under reduced pressure, and the residue wascrystallized from MeOH/EtOAc to give D5 (218 mg, 80%); mp (MeOH/EtOAc);¹H NMR [(CD₃)₂SO] δ 11.13 (s, 1H, NH), 10.44 (s, 1H, NH), 9.88 (br, 2H,NH₂), 8.36 (d, J=7.5 Hz, 2H, ArH), 8.12 (d, J=8.6 Hz, 2H, ArH), 7.86 (d,J=8.8 Hz, 2H, ArH), 7.50 (d, J=8.8 Hz, 2H, ArH), 7.32 (d, J=8.6 Hz, 2H,ArH), 7.32 (d, J=7.5 Hz, 2H, ArH), 7.30 (d, J=8.7 Hz, 2H, ArH), 4.00 (s,3H, N⁺—CH₃); LCMS 319+ve

4-[4-({4-[(1-Methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]quinoliniumdichloride (Cpd. D). Compound D5 (100 mg, 0.28 mmol) was dissolved inEtOH (10 mL) and H₂O (5 mL) by heating. 4-Chloroquinoline (60 mg, 0.36mmol) and c.HCl (3 drops) were added, and the reaction mixture wasrefluxed for 18 h (until TLC with the top phase of 5:4:1 mixture ofn-BuOH:H₂O:CH₃CO₂H showed complete consumption of starting amine). Thereaction mixture was diluted with EtOAc, refluxed for a few minutes, andthen allowed to cool. The resulting precipitate was filtered andcrystallized from MeOH/EtOAc to give Cpd. D (88 mg, 63%); mp (MeOH,EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 14.38 (br, 1H, N⁺H), 11.04 (s, 1H,NH), 10.84 (br, 1H, NH), 10.53 (s, 1H, NH), 8.76 (d, J=8.6 Hz, 1H, ArH),8.58 (d, J=6.7 Hz, 1H, ArH), 8.37 (d, J=7.5 Hz, 2H, ArH), 8.14 (d, J=8.7Hz, 2H, ArH), 8.06-8.01 (m, 4H, ArH), 7.83-7.79 (m 1H, ArH), 7.53-7.48(m, 4H, ArH), 7.33 (d, J=7.6 Hz, 2H, ArH), 6.79 (d, J=6.9 Hz, 1H, ArH),4.00 (s, 3H, N⁺CH₃).

Example E Preparation ofN-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. E)

6-Methyl-N⁴-(4-nitrophenyl)pyrimidine-2,4-diamine (E2). 4-Nitroaniline(BI) [9.22 g, 0.07 mol] and 2-amino-4-chloro-6-methylpyrimidine (EI)(9.30 g, 0.07 mol) were dissolved in 2-ethoxyethanol (330 mL). To theresulting solution were added a few drops of c.HCl, and the resultingmixture was refluxed for 30 min, and then allowed to cool to roomtemperature overnight. After this time, the reaction mixture wasfiltered, and the resulting solid was basified by addition of aqueousammonia solution, and then crystallized from H₂O:EtOH to afford amine E2as an amorphous bright-yellow solid (7.33 g). Concentration of thefiltrate, followed by basification and reprecipitation as before,afforded a further quantity (0.78 g, overall yield 51%). ¹H NMR[(CD₃)₂SO]: 2.14 [s, 3H, ArCH₃], 6.00 [s, 1H, ArNHAr], 6.37 [s, 2H,ArNH₂], 8.00 [ddd, J=10.24, 5.05, 2.96 Hz, 2H, ArH], 8.12 [ddd, J=10.24,4.95, 2.90 Hz, 2H, ArH], 9.75 [s, 1H, ArH]. LCMS (APCI⁺): 246 (100%).

N^(∝)-(4-Aminophenyl)-6-methylpyrimidine-2,4-diamine (E3). To arefluxing suspension of amine E2 (5.46 g, 0.02 mol) in 2:1 EtOH:H₂O (100mL) were sequentially added Fe dust (4.97 g, 0.09 mol) and AcOH (2 mL,2% v/v), and the resulting dark brown suspension was refluxed for ˜14 h.After this time, the hot reaction mixture was filtered through a pad ofCelite, and solvent was removed under reduced pressure. The resultingresidue was extracted with hot water, and the resulting aqueoussuspension filtered through a pad of Celite. Solvent was removed underreduced pressure, and the residue extracted again with hot water. Theresulting aqueous suspension was filtered through a pad of Celite.Solvent was removed to afford amine E3 as a brown-white crystallinesolid (4.85 g, quantitative). ¹H NMR [CD₃)₂SO]: 2.01 [s, 3H, ArCH₃],5.68 [s, 1H, ArNHAr], 5.88 [br s, 2H, ArNH₂], 6.51 [ddd, J=9.67, 4.85,2.94 Hz, 2H, ArH], 7.14 [d, J=8.52 Hz, 2H, ArH], 8.35 [s, 1H, ArH]. LCMS(APCI⁺): 216 (100%).

N-[4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl]-4-nitrobenzamide (E4).4-Nitrobenzoyl chloride (11.00 g, 0.06 mol) and dry pyridine (9.08 mL,0.11 mol) were sequentially added to a solution of amine E3 (4.85 g,0.02 mol) in dry dioxane (200 mL), and the resulting solution heated at˜50° C. for 5 d. After this time, the reaction mixture was cooled toroom temperature, and then basified by addition of aqueous ammoniasolution. The resulting suspension was filtered through a pad of Celiteto afford amide E4 as an amorphous yellow solid (3.40 g, 41%). ¹H NMR[(CD₃)₂SO]: 2.09 [s, 3H, ArCH₃], 5.87 [s, 1H, ArNHAr], 6.08 [br s, 6.08,ArNH₂], 7.68 [m, 4H, ArH], 8.18 [d, J=8.80 Hz, 2H, ArH], 8.36 [d, J=8.80Hz, 2H, ArH], 8.95 [s, 1H, ArH], 10.45 [s, 1H, ArC(O)NHAr]. LCMS(APCI⁺): 365 (100%).

4-Amino-N-[4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]benzamidehydrochloride (E5). To a refluxing suspension of amide E4 (2.10 g, 5.77mmol) in 2:1 EtOH:H₂O (100 mL) were sequentially added Fe dust (1.29 g,23.09 mol) and c.HCl (1-2 mL), and the resulting mixture was refluxedfor 24 h. After this time, the hot reaction mixture was filtered througha pad of Celite, and solvent was removed under reduced pressure. Theresulting residue was crystallized from MeOH:EtOAc to afford amine ES(in two batches) as a cream-coloured amorphous solid (1.94 g, 90%). ¹HNMR [(CD₃)₂SO]): 2.27 [s, 3H, ArCH₃], 6.16 [br s, 2H, ArNH₂], 6.75 [d,J=8.36 Hz, 2H, ArNH₂], 7.74 [m, 8H, ArH], 9.90 [br s, 1H ArH], 10.55 [brs, 1H, ArNHAr], 12.58 [br s, 1H, ArC(O)NH]. LCMS (APCI⁺): 335 (100%).

N-(4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. E). To solution of amine ES (267 mg, 0.720 mmol) in˜1:1:1 MeOH:EtOH:H₂O were sequentially added 4-chloroquinoline (1.06 g,6.48 mmol) and 3 drops of cHCl, and the resulting mixture was refluxedfor ˜24 h. After this time, solvent was removed under reduced pressure,and the resulting residue dried via two MeOH-azeotrope cycles. Theresidue was re-precipitated from MeOH/EtOAc, and then further purifiedby preparative HPLC, to afford Cpd. E as an amorphous yellow solid (16mg, 4%). ¹H NMR [(CD₃)₂SO]: 1.91 [s, 3H, ArCH₃], 6.11 [s, 1H, ArH], 7.02[d, J=6.77 Hz, 1H, ArH], 7.65 [d, J=8.55 Hz, 2H, ArH], 7.71 [br s, 2H,ArNH₂], 7.84 [m, 4H, ArH], 8.04 [m, 2H, ArH], 8.15 [d, J=8.55 Hz, 2H,ArH], 8.63 [d, J=6.77 Hz, 1H, ArH], 8.70 [d, J=8.50 Hz, 1H, ArH], 10.38[s, 2H, ArNHAr & ArH], 10.77 [br s, 1H, ArNHAr], 13.10 [v v br s, 2H,ArC(O)NHAr & quinoline-N⁺H]. LCMS (APCI⁺): 463 (100%). HPLC: 95.7%.

Example F Preparation of1-methyl-4-(3-{[4-(6-nitro-4-quinolinylamino)benzoyl]amino}anilino)pyridiniumchloride (Cpd. F)

Coupling of A7 with 4-chloro-6-nitroquinoline (F1) [Simpson & Wright, J.Chem. Soc., 1948, 1707] as above gave Cpd. F in 97% yield: mp 273-277°C.; ¹H NMR [(CD₃)₂SO] δ 11.33 (br, 1H, NH), 10.70 (s, 1H, NH), 10.60 (s,1H, NH), 8.72-8.69 (m, 2H, ArH), 8.31 (d, J=7.4 Hz, 2H, ArH), 8.24 (d,J=9.3 Hz, 1H, ArH), 8.18 (d, J=8.5 Hz, 2H, ArH), 7.98 (br s, 1H, ArH),7.68-7.65 (m, 3H, ArH), 7.49 (t, J=8.1 Hz, 1H, ArH), 7.21 (d, J=7.5 Hz,2H, ArH), 7.12-7.01 (m, 2H, ArH), 4.02 (s, 3H, N⁺CH₃); HRMS (FAB) calc.for C₂₈H₂₃N₆O₃ (M⁺) m/z 491.1832, found 491.1822.

Example G Preparation of1-methyl-4-(4-(4-(6-nitroquinolin-4-ylamino)benzamido)phenylamino)pyridiniumchloride hydrochloride (Cpd. G)

To a suspension of amide B7 (2.09 g, 5.32 mmol) in dry MeOH (100 mL)were sequentially added 4-chloro-6-nitroquinoline (F1) [1.11 g, 5.32mmol] and a few drops of concentrated HCl, and the resultant mixture wasrefluxed for 12 h. After this time, solvent was removed under reducedpressure, and the residue was re-suspended in 1:1 MeOH:EtOAc. Thismixture was heated to remove MeOH, and then cooled, and the resultingprecipitate collected by filtration. This solid was recrystallized fromMeOH:EtOH to give nitroquinoline Cpd. G (2.48 g, 89%) as an amorphousyellow solid; ¹H NMR [CD₃)₂SO]: δ 3.96 (s, 3H, R₂N⁺⁻CH₃), 7.12 (m, 3H,ArH), 7.35 (d, J=8.81 Hz, 2H, ArH), 7.68 (d, J=8.51 Hz, 2H, ArH), 7.96(d, J=8.81 Hz, 2H ArH), 8.24 (m, 5H, ArH), 8.72 (m, 2H, ArH), 9.83 (d,J=1.69 Hz, 1H, ArH), 10.56 (s, 1H, ArNHAr), 10.67 (s, 1H, ArNHAr), 11.48(br s, 1H, ArNHAr). LCMS (APCI⁺): 492 (20%).

Example H Preparation ofn-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(6-nitroquinolin-4-ylamino)benzamidehydrochloride (Cpd. H)

N-[4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(6-nitroquinolin-4-ylamino)benzamidehydrochloride (Cpd. H). To a solution of amine E5 (304 mg, 0.82 mmol) indry MeOH (30 mL) were sequentially added 4-chloro-6-nitroquinoline (F1)(171 mg, 0.82 mmol) and c.HCl (1-2 drops), and the resulting mixture wasrefluxed for ˜12 h. MS and TLC analysis after this time showed some F1was still present in the reaction mixture, so further aliquots of E5(171 mg, 0.82 mmol) and c.HCl (1-2 drops) were added at 12 h and 24 h.Solvent was then removed from the reaction mixture, and the resultingresidue dissolved in MeOH, and then re-concentrated under reducedpressure. The residue was subjected to a further MeOH azeotrope cycle,and the resulting residue was then reprecipitated from MeOH:Et₂O toafford Cpd. H as an amorphous yellow solid (383 mg, 86%). ¹H NMR (400MHz, DMSO): 2.28 [s, 3H, ArCH₃], 6.18 [br s, 1H, ArH], 7.11 [d, J=6.59Hz, 1H, ArH], 7.65 Hz [d, J=8.52 Hz, 2H, ArH], 7.81 [m, 51-1, ArH &ArNH₂], 8.17 [d, J=8.52 Hz, 2H, ArH], 8.25 [d, J=9.30 Hz, 1H, ArH], 8.69[m, 2H, ArH], 9.77 [s, 1H, ArH], 10.42 [s, 1H, ArH], 10.62 [br s, 1H,ArNHAr], 11.20 [v br s, 1H, ArNHAr], 12.63 [v br s, 1H, ArC(O)NH]. LCMS(APCI⁺): 508 (100%). HPLC: 98.5%.

Example I Preparation of(E)-N-[4-{1-[diaminomethylene]hydrazono)ethyl}phenyl]-4-(6-nitroquinolin-4-ylamino)benzamidehydrochloride (Cpd. I)

(E)-N-[4-(1-{Diaminomethylene}hydrazono)ethyl]-4-nitrobenzenehydrochloride (13). 4-Nitroacetophenone (I1) (49.95 g, 0.30 mol),aminoguanidine sulfate (I2) (51.77 g, 0.20 mol), and c.HCl (10 mL, 0.33mol) were combined in MeOH (600 mL), and the resulting mixture refluxedfor 1 h, and then allowed to cool to room temperature overnight. Afterthis time, solvent was removed under reduced pressure, and the residuecollected by filtration. The resulting solid was washed sequentiallywith MeOH and hexanes to afford diamine I3 as an amorphous white solid(88.19 g, quantitative), which was used without further purification. ¹HNMR [CD₃)₂SO): 2.36 [s, 3H ArC(CH₃)═N—], 7.72 [br s, 4H, ═C(NH₂)₂], 8.23[m. 5H, ArH], 10.42 [v br s, 1.5H]. LCMS (APCI⁺): 222 (100%).

(E)-N-[4-(1-{Diaminomethylene}hydrazono)ethyl]-4-benzaminedihydrochloride (I4). Fe dust (35.00 g, 0.62 mol) and c.HCl (4 mL) weresequentially added to a refluxing suspension of diamine I3 (40.00 g,0.16 mmol) in 2:1 EtOH:H₂O (200 mL), and the resulting yellow suspensionwas refluxed for 14 h. After this time, the hot reaction mixture wasfiltered through a pad of Celite, and solvent was removed under reducedpressure to afford triamine I4 as a dark brown resinous glass (34.93 g,85%). This material was used without further purification. ¹H NMR[CD₃)₂SO): 2.20 [s, 3H ArC(CH₃)═N-], 5.48 [br s, 2H, ArNH₂], 5.54 [d,J=7.98 Hz, 2H, ArH], 7.58 [v br s, 4H, ═C(NH₂)₂], 7.63 [d, J=7.98 Hz,ArH], 10.83 [br s, 1H, ═N⁺(H)—N═]. LCMS (APCI⁺): 192 (100%).

(E)-N-[4-(1-{[Diaminomethylene]hydrazono}ethyl)phenyl]-4-nitrobenzamidehydrochloride (I5). 4-Nitrobenzoyl chloride (16.01 g, 86.26 mmol) wasadded to a solution of triamine I4 (8.59 g, 32.51 mmol) and dry pyridine(13.09 mL, 162.53 mmol) in dry dioxane (300 mL), and the resultingsuspension was refluxed for 2 h, and then allowed to cool roomtemperature overnight. After this time, the reaction mixture wasfiltered to afford a solid which was crystallized from MeOH-EtOAc toafford three batches of amide 15 as an amorphous cream-pale yellow solid(total 3.50 g, 29%). ¹H NMR [CD₃)₂SO): 2.34 [s, 3H, ArC(CH₃)═N—], 7.73[br s, 4H, ═C(NH₂)₂], 7.87 [d, J=8.89 Hz, 2H, ArH], 8.01 [d, J=8.89 Hz,2H, ArH], 8.22 [ddd, J=2.28, 4.27, 9.19 Hz, 2H, ArH], 8.38 [ddd, J=2.28,4.27, 9.19 Hz, 2H, ArH], 10.73 [s, 1H, ═N⁺(H)—N═], 11.02 [s, 1H,ArC(O)NHAr]. LCMS (APCI⁺): 341 (100%).

(E)-N-[4-(1-{[Diaminomethylene]hydrazono}ethyl)phenyl]-4-aminobenzamidedihydrochloride (I6). Fe dust (0.06 g, 1.05 mmol) was added to arefluxing suspension of amide I5 (0.10 g, 0.26 mmol) in 2:1 EtOH/H₂O(100 mL), and the resulting black suspension was refluxed for 1.5 h.After this time, the hot reaction mixture was filtered through a pad ofCelite, and solvent was removed under reduced pressure. The residue wasdried via two MeOH-azeotrope cycles, and then re-dissolved in MeOH. Thissolution was acidified with a few drops of methanolic HCl, and thendiluted with EtOAc. The resulting suspension was filtered to afford anamorphous tan solid, which was re-dissolved in MeOH. Solvent was removedunder reduced pressure, and the residue re-dissolved in hot H₂O. Theresulting suspension was filtered through a pad of Celite (with a smallvolume of cold MeOH), and solvent was removed under reduced pressure.The residue was re-precipitated from MeOH-EtOAc to afford amine I6 as anamorphous yellow solid (0.36 g, quantitative). ¹H NMR [CD₃)₂SO): 2.25[s, 3H, ArC(CH₃)═N—], 5.74 [s, 2H, ArNH₂], 6.40 {v br s, 4H, ═C(NH₂)₂],6.60 [d, J=8.66 Hz, 2H, ArH], 7.77 [m, 6H, ArH], 9.81 [s, 1H,ArC(O)NHAr].

(E)-N-[4-(1-([Diaminomethylene]hydrazono)ethyl}phenyl]-4-(6-nitroquinolin-4-ylamino)benzamidehydrochloride (Cpd. I). To a suspension of amine I6 (˜100 mg based onI5, ˜0.26 mmol) in dry EtOH (60 mL) were sequentially added4-chloro-6-nitroquinoline (220 mg, 1.04 mmol) and c.HCl (3 drops), andthe resulting mixture refluxed for 64 h. After this time, the reactionmixture was filtered to afford a yellow solid, which was crystallizedfrom MeOH—HCl:EtOAc to afford Cpd. I as an amorphous bright yellow solid(113 mg, 84%). ¹H NMR [CD₃)₂SO): 2.36 [s, 3H, ArC(CH₃)═N—], 7.09 [d,J=6.95 Hz, 1H ArH], 7.69 [d, J=8.62 Hz, ArH], 7.77 [br s, 4H, ═C(NH₂)₂],7.91 [d, J=8.92 Hz, 2H, ArH], 8.00 [d, J=8.92 Hz, 2H, ArH], 8.21 [d,J=8.62 Hz, 2H, ArH], 8.31 [d, J=9.32 Hz, 214, ArH], 8.70 [d, J=6.95 Hz,1H, ArH], 8.74 [dd, J=9.32, 2.29 Hz, 1H, ArH], 9.86 [s, 1H, ArH], 10.55[s, 1H, ArNHAr], 11.41 [s, 1H, ArC(O)NHAr], 11.60 [br s, 1H, 1H,═N+(H)—N═]. LCMS (APCI⁺): 484 (100%). HPLC: 97.1%.

Example J Preparation of4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]-6-nitroquinoliniumdichloride (Cpd. J)

Compound C6 (100 mg, 0.28 mmol) was dissolved in EtOH (10 mL) and H₂O (5mL) by heating. 4-Chloro-6-nitroquinoline (F1) (75 mg, 0.36 mmol) and c.HCl (3 drops) were added, and the reaction mixture was refluxed for 18h. (until TLC with the top phase of 5:4:1 mixture of n-BuOH:H₂O:AcOHshowed complete consumption of starting amine). The reaction mixture wasdiluted with EtOAc, refluxed for a few minutes, and allowed to cool. Theresulting precipitate was filtered and crystallized from MeOH/EtOAc togive Cpd. J (148 mg, 94%); mp (MeOH, EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO]:δ14.90 (br 1H, N^(+H),) 11.36 (br, 1H, NH), 10.93 (s, 1H, NH), 10.65 (s,1H, NH), 9.81 (d, J=2.2 Hz, 1H, H-5), 8.71 (dd, J=9.3, 2.3 Hz, 1H, H-7),8.60 (d, J=7.0 Hz, 1H, H-8), 8.33 (d, J=7.5 Hz, 2H, py H-2, 6), 8.22 (d,J=9.5 Hz, 1H, H-2), 8.02 (d, J=8.9 Hz, 2H, ArH), 7.81-7.92 (m, 2H, ArH),7.68 (t, J=7.8 Hz, 1H, ArH), 7.58 (dd, J=8.6, 1.3 Hz, 1H, ArH), 7.50 (d,J=7.0 Hz, 2H, ArH), 7.28 (d, J=7.5 Hz, 2H, ArH), 6.91 (d, J=7.0 Hz, 1H,ArH), 3.98 (s, 3H, N⁺CH₃).

Example K Preparation of4-[3-({4-[(6-ammonio-4-quinolinyl)amino]benzoyl}amino)anilino]-1-methylpyridiniumdichloride (Cpd. K)

To a suspension of Cpd. F (200 mg, 0.41 mmol) in ˜6:1 EtOH:H₂O (6 mL)was added Fe dust (200 mg), and the resulting suspension was refluxedfor 3 h. The hot reaction mixture was filtered through a pad of Celite,and the Celite pad washed with hot EtOH. The EtOH extracts were combinedand evaporated to dryness, and the residue extracted into water. Thesolution was filtered through Celite, and then evaporated to dryness.The residue was azeotroped with methanol (3×30 mL), and then dissolvedin MeOH (20 mL). Methanolic HCl (1.25 M, 1 mL) was added, and thesolution stirred for 30 min. The solution was evaporated to dryness, andthe residue crystallized from MeOH/EtOAc to give Cpd. K (160 mg, 80%):mp, (MeOH/EtOAc) 270-274° C.; ¹H NMR [(CD₃)₂SO] δ 14.50 (br, 1H, N⁺H),10.80 (s, 1H, NH), 10.57 (s, 1H, NH), 10.36 (s, 1H, NH), 8.31 (d, J=7.5Hz, 3H, ArH), 8.15 (d, J=8.6 Hz, 2H, ArH), 7.99 (t, J=1.9 Hz, 1H, ArH),7.85 (d, J=9.1 Hz, 1H, ArH), 7.68 (br d, J=9.2 Hz, 1H, ArH), 7.62 (d,J=8.6 Hz, 2H, ArH), 7.51-7.12 (m, 3H, ArH), 7.23 (d, J=7.6 Hz, 2H, ArH),7.09 (dd, J=7.9, 2.8 Hz, 1H, ArH), 6.95 (d, J=6.7 Hz, 1H, ArH), 3.96 (s,3H, N⁺CH₃), the signal for NH₂ was not observed. HRMS (FAB⁺) calc forC₂₈H₂₅N₆O (M⁺¹) m/z 461.2090, found 461.2108; Anal. calc.CHN forC₂₈H₂₇N₆Cl₃O.0.25H₂O: C, 58.6; H, 4.8; N, 14.6; found, C, 58.5; H, 4.8;N, 14.5%.

Example L Preparation of4-[4-({4-[(6-ammonio-4-quinolinyl)amino]benzoyl}amino)anilino]-1-methylpyridiniumdichloride (Cpd. L)

To a refluxing suspension of Cpd. G (150 mg, 0.28 mmol) in 2:1 EtOH:F120(50 mL) was added Fe dust (60 mg, 1.13 mmol), and the resulting mixturewas refluxed overnight. After this time, the hot reaction mixture wasfiltered through a pad of Celite, and solvent was again removed underreduced pressure. The residue was re-dissolved in MeOH, and solvent wasremoved under reduced pressure. This latter process was repeated twicemore, and the residue was then recrystallized from HCl-MeOH: Et₂O togive Cpd. L (33 mg, 22%) as an amorphous yellow-brown solid. ¹H NMR[(CD₃)₂SO]: δ 3.95 (s, 3H, R₂N⁺⁻CH₃), 6.94 (d, J=6.67 Hz, 1H, ArH), 7.13(d, J=7.36 Hz, 2H, ArH), 7.34 (d, J=8.85 Hz, 2H, ArH), 7.43 (dd, J=9.06,2.17 Hz, 1H, ArH), 7.45 (d, J=1.97 Hz, 1H, ArH), 7.62 (d, J=8.61 Hz, 2H,ArH), 7.85 (d, J=9.06 Hz, 1H, ArH), 7.95 (d, J=8.86 Hz, 1H, ArH), 8.15(d, J=8.59 Hz, 2H, ArH), 8.26 (d, J=7.39 Hz, 2H, ArH), 8.32 (t, J=6.11Hz, 1H, ArH), 10.35 (s, 1H, ArNHAr), 10.51 (s, 1H, ArNHAr), 10.69 (s,1H, ArNHAr), 14.49 (br s, 1H, quinolone-N⁺—H) [NH₂ signal not observed].LCMS (APCI⁺): 462 (100%). HPLC: 97%.

Example M Preparation of6-amino-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]quinoliniumdichloride (Cpd. M)

To a vigorously stirred suspension of compound Cpd. J (80 mg, 0.15 mmol)in 5:1 EtOH/H₂O (5 mL) was added Fe dust (43 mg), and the mixture wasbrought to reflux. Two drops of c.HCl were then added, and refluxingcontinued for a further 2 h (until TLC with the top phase of 5:4:1mixture of n-BuOH:H₂H:AcOH showed complete consumption of startingmaterial). The reaction mixture was diluted with EtOH (100 mL) andbrought to reflux, and the hot mixture was filtered through a pad ofCelite. The top residue of the Celite pad was extracted three furthertimes with hot EtOH to ensure the complete extraction of the amine. TheEtOH fractions were combined and evaporated to dryness, and the residuewas extracted with hot water. The solution was evaporated to dryness,and azeotroped several times with EtOH. The resulting residue wasdissolved in a small amount of MeOH, methanolic HCl (1.25 M, 1 mL) wasadded, and the solution was stirred for 10 min. The solution was thendiluted with EtOAc, and then some of the MeOH was evaporated. Theresulting precipitate was filtered and crystallized from MeOH/EtOAc togive Cpd. M (56 mg, 70%); mp>310° C. ¹H NMR [(CD₃)₂SO] δ 14.5 (br, 1H,N⁺H), 11.22 (s, 1H, NH), 10.59 (s, 1H, NH), 9.55 (br, 1H, NH), 8.32 (d,J=7.2 Hz, 2H, ArH), 8.18 (d, J=6.1 Hz, 1H, ArH), 7.95-7.91 (m, 4H, ArH),7.76 (d, J=8.8 Hz, 1H, ArH), 7.66 (t, J=5.6 Hz, 1H, ArH), 8.07 (d, J=8.4Hz, 1H, ArH), 7.40-7.37 (m, 3H, ArH), 7.33-7.29 (m, 3H, ArH), 6.71 (d,J=6.2 Hz, 1H, ArH), 5.75 (br S, 2H, NH₂), 3.98 (s, 3H, N⁺CH₃).

Example N Preparation of4-[4-({4-[(6-amino-4-quinolinyl)amino]anilino}carbonyl)anilino]-1-methylpyridiniumchloride (Cpd. N)

1-Methyl-4-[4-({4-[(6-nitro-4-quinolinyl)amino]anilino}carbonyl)-anilino]pyridiniumchloride (N1). To a suspension of 4-chloro-6-nitroquinoline (70 mg, 0.33mmol) in MeOH (10 mL) was added compound D5 (100 mg, 0.28 mmol), and themixture was stirred at 20° C. for 1 h (until a clear solution obtained).A drop of c. HCl was then added, and refluxing was continued for afurther 20 h. TLC analysis (eluting with the top phase of a 5:4:1mixture of n-BuOH:H₂O:AcOH) showed some D5 remained, so more4-chloro-6-nitroquinoline (35 mg, 0.16 mmol) was added and refluxing wascontinued for a further 4 h. The reaction mixture was then diluted withEtOAc and the resulted precipitate was filtered and washed sequentiallywith EtOAc, CH₂Cl₂ and diisopropylether, and finally crystallized fromMeOH/EtOAc to give Cpd. N1 (111 mg, 75%), mp (MeOH/EtOAc)>300° C.; ¹HNMR [(CD₃)₂SO] δ 14.50 (br, 1H, N⁺H); 11.42, (br s, 1H, NH), 11.10 (s,1H, NH), 10.58 (s, 1H, NH), 9.82 (d, J=2.1 Hz, 1H, ArH), 8.72 (dd,J=9.3, 2.2 Hz, 1H, ArH), 8.60 (d, J=7.0 Hz, 1H, ArH), 8.42 (d, J=7.4 Hz,2H, ArH), 8.24 (d, J=8.6 Hz, 1H, ArH), 8.15 (d, J=8.6 Hz, 2H, ArH), 8.04(d, J=8.8 Hz, 2H, ArH), 7.51 (t, J=8.3 Hz, 4H, ArH), 7.34 (d, J=7.5 Hz,2H, ArH), 6.91 (d, J=7.0 Hz, 1H, ArH), 4.01 (s, 3H, N⁺CH₃); LCMS 489+ve.

4-[4-({4-[(6-Amino-4-quinolinyl)amino]anilino}carbonyl)anilino]-1-methylpyridiniumchloride (Cpd. N). To a solution of compound N1 (102 mg, 0.19 mmol) in˜5:1 EtOH:H₂O (3 mL) was added Fe dust (53 mg) and a drop of AcOH, andthe resulting suspension vigorously stirred and refluxed for 3 h. Thehot reaction mixture was filtered through Celite, and the Celite padwashed with more hot EtOH. The EtOH fractions were combined andevaporated to dryness, and the residue extracted into hot water. Thissolution was filtered through Celite and then evaporated to dryness. Theresidue was azeotroped with MeOH (3×20 mL), and then dissolved in MeOH(10 mL). Methanolic HCl (1.25 M, 2 mL) was added, and the solutionstirred for 10 min. The solvent was evaporated to dryness, and theresidue crystallized with MeOH/EtOAc to give Cpd. N (92 mg, 96%) mp(MeOH/EtOAc) ¹H NMR (CD₃)₂SO] δ 14.17 (d, J=5.8 Hz, 1H NH), 11.11 (s,1H, NH), 10.52 (s, 1H, NH), 10.21 (s, 1H, NH), 8.37 (d, J=7.5 Hz, 2H,ArH), 8.22 (t, J=6.6 Hz, 1H, ArH), 8.14 (d, J=8.6 Hz, 2H, ArH), 7.98 (d,J=8.9 Hz, 2H, ArH), 7.79 (d, J=9.1 Hz, 1H, ArH), 7.52-7.59 (m, 3H, ArH),7.44-7.40 (m, 3H, ArH), 7.43 (d, J=7.6 Hz, 2H, ArH), 6.67 (d, J=6.8 Hz,1H, ArH), 4.01 (s, 3H, N⁺CH₃) partial signal for the N⁺H₃ was observed.LCMS 461+ve.

Example O Preparation of4-[4-({4-[(6-dimethylammonio-4-quinolinyl)amino]benzoyl}amino)-anilino]-1-methylpyridiniumdichloride (Cpd. O)

To a solution of 6-amino-4-quinolone (O1) (112 mg, 0.71 mmol) andaqueous formaldehyde (40% w/v, 1.6 mL, 21.2 mmol) in EtOH (10 mL) weresequentially added NaBH₃CN (335 mg, 5.67 mmol) and 1N HCl (2.8 mL, 2.8mmol), and the resulting bright yellow suspension was stirred at roomtemperature for 15 minutes. After this time, solvent was removed underreduced pressure, and the residue then dried by two MeOH azeotropecycles. The residue was resuspended in MeOH and filtered through Celite,and solvent was removed under reduced pressure. The residue wascrystallized from MeOH:EtOAc to give 6-dimethylamino-4-quinolone (O2)(80 mg, 60%) as an amorphous tan solid; ¹H NMR [(CD₃)₂SO] δ 2.93 [s, 6H,ArN(CH₃)₂], 5.90 [d, J=7.00 Hz, 1H, ArH], 7.13 [br s, ArOH], 7.24 [m,2H, ArH], 7.45 [dd, J=7.41, 2.29 Hz, 1H, ArH], 7.74 [d, J=7.00 Hz, 1H,ArH]. LCMS (APCI⁺): 189 (100%). R_(f)=0.48 (10% MeOH:CH₂Cl₂).

Quinolone O2 (80 mg, 0.43 mmol) was refluxed in POCl₃ (10 mL) for 1 h.After this time, excess POCl₃ was removed under reduced pressure, andthe residue was dissolved in CH₂Cl₂, cooled to 0° C., and treated withaqueous ammonia. The resulting mixture was extracted with CH₂Cl₂ (×3).The combined organic extracts were washed sequentially with H₂O (×1) andbrine (×1), and then dried over MgSO₄. Solvent was removed under reducedpressure to give 6-dimethylamino-4-chloroquinoline (O3) (70 mg, 80%) asa bright yellow oil [Riegel et al., J. Am. Chem. Soc., 1946, 68, 1264].¹H NMR [(CD₃)₂SO] δ 3.10 [s, 6H, ArN(CH₃)₂], 6.96 [d, J=2.85 Hz, 1H,ArH], 7.74 [dd, J=9.36, 2.85 Hz, 1H, ArH], 7.57 [d, J=4.69 Hz, 1H, ArH],7.90 [d, J=9.36 Hz, 1H, ArH], 8.46 Hz [d, J=4.69 Hz, 1H, ArH]. LCMS(APCI⁺): 207 (100%), 209 (40%). R_(f)=0.73 (10% MeOH:CH₂Cl₂).

To a solution of amine B7 (260 mg, 0.65 mmol) dissolved in 1:2 EtOH:H₂O(6 mL) were sequentially added cHCl (0.19 mL, 6.34 mmol) and 03 (140 mg,0.70 mmol). The resulting mixture was refluxed for 72 h (reactionprogress followed by TLC, eluting with the top phase of a 5:4:1 mixtureof n-BuOH:H₂O:AcOH; R_(f)=0.30-0.40, bright yellow spot at 365 nm).After this time, solvent was removed under reduced pressure. The residuewas re-dissolved in MeOH, and solvent was again removed under reducedpressure. This latter process was repeated once more, and the residuewas crystallized twice from MeOH:EtOAc to afford Cpd. O (163 mg, 45%) asan amorphous yellow solid. ¹H NMR [(CD₃)₂SO] δ 3.06 (s, 6H, Ar(CH₃)₂),3.93 (s, 3H, R₂N⁺⁻CH₃), 7.07 (d, J=6.58 Hz, 2H, ArH), 7.12 (d, J=4.98Hz, 1H, ArH), 7.22 (d, J=2.43 Hz, 1H, ArH), 7.30 (d, J=8.70 Hz, 2H,ArH), 7.42 (m, 3H, ArH), 7.78 (d, J=9.28 Hz, ArH), 7.92 (d, J=10.75 Hz,2H, ArH), 8.01 (d, J=8.64 Hz, 1H, ArH), 8.19 (d, J=6.93 Hz, 2H, ArH),8.32 (d, J=4.90 Hz, 1H, ArH), 8.50 (br s, 1H, —NH—), 8.91 (s, 1H, —NH—),10.28 (s, 1H, —NH—), 10.80 (v br s, 1H, —NH—). LCMS (APCI⁺): 490 (100%).HPLC: 95%.

Example P Preparation ofN-[4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(6-(dimethylamino)quinolin-4-ylamino)benzamidehydrochloride (Cpd. P)

To a solution of amide ES (228 mg, 0.62 mmol) in 1:2 EtOH:H₂O (20 mL)were sequentially added c.HCl (0.17 mL, 5.61 mmol) and4-chloro-6-dimethylaminoquinoline (03) (140 mg, 0.68 mmol), and theresulting mixture was refluxed for ˜12 h. MS and TLC analysis (elutingwith the top phase of a 5:4:1 mixture of n-BuOH:H₂O:AcOH) after thistime showed some starting material still present in the reactionmixture, thus more O3 (140 mg, 0.68 mmol) was added. After a further fewhours refluxing, TLC showed the reaction was complete, and thus solventwas removed under reduced pressure. The residue was crystallized fromMeOH:EtOAc to afford Cpd. P as an (extremely fine) amorphous yellowsolid (170 mg, 51%). ¹H NMR [(CD₃)₂SO] δ 2.28 [s, 3H], [s, 6H,ArN(CH₃)₂], 6.21 [br s, 1H, ArH], 6.89 [d, J=6.71 Hz, 1H, ArH], 7.65 [m,5H, ArH], 7.82 [m, 4H, ArH & ArNH₂], 7.96 [d, J=9.39 Hz, 1H, ArH], 8.18[d, J=8.57 Hz, 2H, ArH], 8.35 [t, J=12.38, 6.19 Hz, 1H, ArH], 10.44 [brs, 1H, ArH], 10.65 [br s, 1H, ArNHAr], 10.71 [s, 1H, ArNHAr], 12.75 [brs, 1H, ArC(O)NH], 14.56 [d, J=4.10 Hz, 1H, quinolinyl N⁺H]. LCMS(APCI⁺): 506 (100%). HPLC: 97.6%.

Example Q Preparation of6-(dimethylamino)-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]anilino}carbonyl)anilino]quinoliniumdichloride (Cpd. Q)

Amine A7 (100 mg, 0.28 mmol) was dissolved in EtOH (20 mL) and H₂O (10mL) by heating. 4-Chloro-6-dimethylaminoquinoline (03) (70 mg, 0.33mmol) and c.HCl (3 drops) were then added, and the mixture was refluxedfor 3 d. (until TLC with the top phase of 5:4:1 mixture ofn-BuOH:H₂O:AcOH showed complete consumption of starting amine). Thereaction mixture was evaporated to dryness, and the residue azeotropedwith EtOH. The residue was dissolved in a small volume of MeOH, whichwas then diluted with EtOAc. The resulting precipitate was filtered andcrystallized from MeOH/EtOAc to give 163 mg of crude product, which waspurified by preparative HPLC to give Cpd. Q (70 mg, 45%); mp(MeOH/EtOAc), >300° C.; ¹H NMR [(CD₃)₂SO] δ 14.38 (br, 1 H, N⁺H), 10.71(s, 1H, NH), 10.56 (s, 1H, NH), 10.52 (s, 1H, NH), 8.36 (d, J=6.7 Hz,1H, ArH), 8.31 (d, J=7.3 Hz, 2H, ArH), 8.18 (d, J=8.6 Hz, 2H, ArH), 7.99(brs, 1H, ArH), 7.92 (d, J=9.4 Hz, 1H, ArH), 7.69-7.64 (m, 4H, ArH),7.54 (d, J=2.3 Hz, 1H, ArH), 7.49 (t, J=8.1 Hz, 1H, ArH), 7.21 (d, J=7.4Hz, 2H, ArH), 7.08 (d, J=7.9 Hz, 1H, ArH), 6.91 (d, J=6.7 Hz, 1H, ArH),3.98 (s, 3H, N⁺CH₃), 3.14 [s, 6H, N(CH₃)₂].

Example R Preparation of1-methyl-4-[3-({4-[(7-nitro-4-quinolinyl)amino]benzoyl}amino)anilino]pyridiniumchloride (Cpd. R)

Coupling of A7 with 4-chloro-7-nitro quinoline (Ruchelman et al. Biorg.Med. Chem. 2004, 12, 3731) gave Cpd. R in 89% yield; mp (MeOH/EtOAC),302-306° C. (dec); ¹H NMR [(CD₃)₂SO] δ 11.50 (br, 1H, NH), 10.74 (s, 1H,NH), 10.58 (s, 1H, NH), 9.04 (d, J=9.3 Hz, 1H, ArH), 8.90 (d, J=2.3 Hz,1H, ArH), 8.77 (d, J=6.7 Hz, 1H, ArH), 8.51 (dd, J=9.3, 4.5 Hz, 1H,ArH), 8.31 (d, J=7.4 Hz, 2H, ArH), 8.18 (d, J=8.6 Hz, 2H, ArH), 7.98 (t,J=1.9 Hz, 1H, ArH), 7.70 (d, J=8.6 Hz, 3H, ArH), 7.49 (t, J=8.1 Hz, 1H,ArH), 7.22 (d, J=7.6 Hz, 2H, ArH), 7.15 (d, J=6.7 Hz, 1H, ArH), 7.09 (brd, J=7.8 Hz, 1H, ArH), 3.96 (s, 3H, N⁺CH₃); HRMS (FAB⁺) calc. forC₂₈H₂₄N₆O₃ (M⁺¹) m/z 491.1832, found 491.1825.

Example S Preparation of1-methyl-4-[4-(4-{7-nitroquinolin-4-ylamino}benzamido)-phenylamino]pyridiniumdichloride (Cpd. S)

To a solution of amine B7 (1.46 g, 3.73 mmol) in dry MeOH (20 mL) weresequentially added 4-chloro-7-nitroquinoline (S1) (0.78 g, 3.73 mmol)and two drops of c.HCl, and the resulting mixture was refluxed for 4 h.After this time, the temperature was reduced to −40° C. and heating wascontinued for a further 65 h. After this time, solvent was removed underreduced pressure. The residue was re-dissolved in MeOH, and solvent wasagain removed under reduced pressure. This latter process was repeatedonce more, and the residue was then re-precipitated twice fromMeOH:Et₂O. Finally, a portion of the solid thus obtained wasre-precipitated from MeOH:EtOAc to afford Cpd. S as an amorphousyellow-orange solid (0.070 g, 3%). ¹H NMR [(CD₃)₂SO]: 3.96 [s, 3H,R₂N⁺CH₃], 7.14 [m, 3H, ArH], 7.35 [d, J=8.85 Hz, 2H, ArH], 7.68 [d,J=8.57 Hz, 2H, ArH], 7.96 [d, J=8.85 Hz, 2H, ArH], 8.18 [d, J=8.57 Hz,2H, ArH], 8.26 [d, J=7.37 Hz, 2H, ArH], 8.50 [dd, J=9.28, 2.23 Hz, 1H,ArH], 8.77 [d, J=6.65 Hz, 1H, ArH], 8.94 [d, J=2.23 Hz, 1H, ArH], 9.08[d, J=9.28 Hz, 1H, ArH], 10.55 [s, 1H, ArNHAr], 10.71 [s, 1H, ArNHAr],11.21 [br s, 1H, ArC(O)NHAr], 11.21 [v v br s, 1H, quinoline-N⁺H]; LCMS(APCI⁺): 492 (100%), 493 (30%). HPLC: 98.1%.

Example T Preparation of4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]-7-nitroquinoliniumdichloride (Cpd. T)

Compound C6 (92 mg, 0.25 mmol) was dissolved in EtOH (10 mL) and H₂O (5mL) by heating. 4-Chloro-7-nitroquinoline (65 mg, 0.31 mmol) and c. HCl(3 drops) were then added and the mixture was refluxed for 18 h. (untilTLC with the top phase of 5:4:1 mixture of n-BuOH:H₂O:AcOH showedcomplete consumption of starting amine). The reaction mixture was thendiluted with EtOAc, refluxed for a few minutes, and allowed to cool. Theresulting precipitate was filtered and crystallized twice fromMeOH/EtOAc to give Cpd. T (104 mg, 71%); mp (MeOH, EtOAc)>300° C.; ¹HNMR [(CD₃)₂SO] δ14.80 (br 1H, N⁺H), 10.89 (br, 2H, 2×NH), 10.60 (s, 1H,NH), 8.96 (d, J=9.0 Hz, 1H, ArH), 8.84 (bs, 1H, ArH), 8.67 (d, J=6.5 Hz,1H, ArH), 8.32 (d, J=7.3 Hz, 2H, ArH), 8.0-7.91 (m, 4H, ArH), 7.68 (t,J=7.8 Hz, 1H, Aril), 7.57 (d, J=9.4 Hz, 1H, ArH), 7.49 (d, J=8.7 Hz, 2H,ArH), 7.29 (d, J=7.4 Hz, 2H, ArH), 6.94 (d, J=6.6 Hz, 1H, ArH), 3.99 (s,3H, N⁺CH₃). HRMS (FAB⁺) calc. for C₂₈H₂₃N₆O₃ (M⁺¹) m/z 491.1832, found491.1830.

Example U Preparation of4-[4-({4-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]-7-nitroquinoliniumdichloride (Cpd. U)

Compound D5 (100 mg, 0.28 mmol) was dissolved in EtOH (20 mL) and H₂O(10 mL) by heating. 4-Chloro-7-nitroquinoline (70 mg, 0.33 mmol) andc.HCl (3 drops) were then added and the reaction mixture was refluxedfor 18 h. (until TLC with the top phase of 5:4:1 mixture ofn-BuOH:H₂O:AcOH showed complete consumption of starting amine). Thereaction mixture was then diluted with EtOAc, refluxed for a fewminutes, and allowed to cool. The resulting precipitate was filtered andcrystallized twice from MeOH/EtOAc to give Cpd. U (99 mg, 63%); mp(MeOH/EtOAc) 268° C. (dec); ¹H NMR [(CD₃)₂SO] δ 10.93 (brs, 2H, 2×NH),10.53 (s, 1H, NH), 8.94 (d, J=9.2 Hz, 1H, ArH), 8.85 (bs, 1H, ArH), 8.67(d, J=6.7 Hz, 1H, ArH), 8.49 (d, J=9.2 Hz, 1H, ArH), 8.37 (d, J=7.5 Hz,2H, ArH), 8.13 (d, J=8.6 Hz, 2H, ArH), 8.51 (d, J=8.8 Hz, 2H, ArH), 7.50(t, J=8.7 Hz, 4 H, ArH), 7.31 (d, J=7.5 Hz, 2H, ArH), 6.94 (d, J=6.8 Hz,1H, ArH), 4.02 (s, 3H, N⁺CH₃). APCI⁺ve 461.

Example V Preparation of preparation of4-[3-({4-[(7-ammonio-4-quinolinyl)amino]benzoyl}amino)anilino]-1-methylpyridiniumdichloride (Cpd. V)

Fe dust reduction of Cpd. R as above gave Cpd. V in 83% yield: mp281-285° C.; ¹H NMR [(CD₃)₂SO] δ 13.73 (br, 1H, NH), 10.79 (s, 1H, NH),10.56 (s, 1H, NH), 10.53 (br s, 1H, NH), 8.41 (d, J=9.3 Hz, 1H, ArH),8.31 (d, J=7.4 Hz, 2H, ArH), 8.24 (d, J=7.0 Hz, 1H, ArH), 8.14 (d, J=8.6Hz, 2H, ArH), 7.98 (t, J=1.9 Hz, 1H, ArH), 7.67 (br d, J=9.2 Hz, 1H,ArH), 7.59 (d, J=8.6 Hz, 2H, ArH), 7.48 (t, J=8.1 Hz, 1H, ArH), 7.23 (d,J=7.5 Hz, 2H, ArH), 7.10-7.06 (m, 2H, ArH), 6.86 (d, J=2.1 Hz, 1H, ArH),6.76 (br s, 2H, NH₂), 6.6.8 (d, J=7.0 Hz, 1H, ArH), 3.98 (s, 3H, N⁺CH₃);HRMS (FAB⁺) calc. for C₂₈H₂₅N₆O (M⁺) m/z 461.2090, found 461.2108; Anal.calc. for C₂₈H₂₆N₆ClO₃. 1.25H₂O: C, 60.5; H, 5.2; N, 15.1; Cl, 12.8;found, C, 60.6; H, 5.2; N, 15.0; Cl, 13.0%.

Example W Preparation of1-methyl-4-[4-(4-{7-aminoquinolin-4-ylamino}benzamido)-phenylamino]pyridiniumdichloride (Cpd. W)

To a refluxing suspension of Cpd. S (40 mg, 0.07 mmol) in 2:1 EtOH:H₂O(50 mL) was added Fe dust (15 mg, 0.28 mmol), and the resulting mixturewas refluxed for a few hours until complete. After this time, the hotreaction mixture was filtered through a pad of Celite, and solvent wasremoved under reduced pressure. The residue was dried via threeMeOH-azeotrope cycles, and finally recrystallized from HCl-MeOH:EtOAc togive Cpd. W (34 mg, 90%) as an amorphous yellow solid; mp (MeOH,EtOAc)>280° C.; ¹H NMR [(CD₃)₂SO]: 3.95 (s, 3H, R₂N⁺CH₃), 6.67 (d,J=7.00 Hz, 1H, ArH), 6.92 (d, J=2.13 Hz, 1H, ArH), 7.07 (dd, J=9.38,2.13 Hz, 1H, ArH), 7.18 (br d, J=6.45 Hz, 2H, ArNH₂), 7.34 (d, J=8.89Hz, 2H, ArH), 7.60 (d, J=8.60 Hz, 2H, ArH), 7.97 (d, J=, 8.89 Hz, ArH),8.16 (d, J=8.60 Hz, 2H ArH), 8.22 (t, J=6.66 Hz, 1H, ArH), 8.27 (d,J=7.49 Hz, 2H, ArH), 8.51 (d, J=9.39 Hz, 1H, ArH), 10.58 (s, 1H,ArNHAr), 10.71 (s, 1H, ArNHAr), 11.00 (s, 1H, ArC(O)NHAr), 14.08 (d,J=6.09 Hz, 1H, quinoline-N⁺—H); LCMS (APCI⁺): 462 (100%); HPLC: 96.1%.

Example X Preparation of7-amino-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]quinoliniumdichloride (Cpd. X)

To a vigorously stirred suspension of compound Cpd. T (107 mg, 0.19mmol) in 5:1 EtOH:H₂O (5 mL) was added Fe dust (43 mg), and the mixturewas brought to reflux. Two drops of c.HCl were added, and refluxing wascontinued for 2 h (until TLC with the top phase of 5:4:1 mixture ofn-BuOH:H₂O:AcOH showed complete consumption of starting material). Thereaction mixture was then diluted with EtOH (100 mL), and brought toreflux. The hot mixture was filtered through a pad of Celite, and thetop layer of the Celite pad extracted three times with hot EtOH toensure complete extraction of the amine. The combined ethanol extractswere evaporated to dryness, and the residue was extracted with hot H₂O.The solution was evaporated to dryness, and azeotroped several timeswith EtOH. The resulting residue was dissolved in a small volume ofMeOH, methanolic HCl was added (1.25 M, 1 mL), and the solution wasstirred for 10 min. The solution was diluted with EtOAc, and some of theMeOH was evaporated. The resulting precipitate was filtered andcrystallized from MeOH/EtOAc to give Cpd. X (99 mg, 98%); mp(MeOH/EtOAc>300° C.; ¹H NMR [(CD₃)₂SO] δ 13.48 (s, 1 H, N⁺H), 10.98 (s,1H, NH), 10.59 (s, 1H, NH), 10.32 (s, 1H, NH), 8.36-8.31 (m, 3H, ArH),8.14 (d, J=7.0 Hz, 1H, ArH), 8.00-7.91 (m, 4H, ArH), 7.67 (t, J=7.7 Hz,1H, ArH), 7.57 (d, J=7.4H, 1H, ArH), 7.40 (d, J=8.6 Hz, 2H, ArH), 7.02(d, J=9.2 Hz, 1H, ArH), 6.82 (bs, 1H, ArH), 6.66 (brs, 2H, NH₂), 6.43(d; J=6.9 Hz, 1H, ArH), 3.99 (s, 3H, N⁺CH₃). HRMS (FAB) calc. forC₂₈H₂₅N₆O (M⁻¹) m/z 461.2090, found 461.2085.

Example Y Preparation of4-[4-({4-[(7-amino-4-quinolinyl)amino]anilino}carbonyl)anilino]-1-methylpyridiniumchloride (Cpd. Y)

To a suspension of Cpd. U (101 mg, 0.19 mmol) in ˜5:1 EtOH:H₂O (3 mL)was added Fe dust (53 mg), followed by a drop of AcOH. The resultingsuspension was vigorously stirred and refluxed for 3 h. The hot reactionmixture was filtered through Celite, and the Celite pad was washed withhot EtOH. The combined EtOH extracts were evaporated to dryness and theresidue extracted into hot water. The solution was filtered throughCelite, and then evaporated to dryness. The residue was azeotroped withMeOH (3×20 mL), and then dissolved in MeOH (10 mL). Methanolic HCl (1.25M, 2 mL) was added, and the solution stirred for 10 min. The solutionwas then evaporated to dryness, and the residue crystallized fromMeOH/EtOAc to give compound Cpd. V (55 mg, 58%) mp (MeOH/EtOAc) 290-295°C.; ¹H NMR (CD₃)₂SO] δ 13.44 (br, 1H, N⁺H), 11.04 (s, 1H, NH), 10.50 (s,1 H, NH), 10.31 (s, 1H, NH), 8.38-8.33 (m, 3H, ArH), 8.15-8.11 (m, 3H,ArH), 7.97 (d, J=8.9 Hz, 2H, ArH), 7.51 (d, J=8.7 Hz, 2H, ArH), 7.41 (d,J=9.9 Hz, 2H, ArH), 7.33 (d, J=7.5 Hz, 2H, ArH), 7.03 (dd, J=9.2, 2.1Hz, 1H, ArH), 6.81 (d, J=2.2 Hz, 1H, ArH), 6.67 (bs, 2H, NH₂), 6.44 (d,J=7.1 Hz, 1H, ArH), 4.01 (s, 3H, N⁺CH₃); LCMS 461+ve.

Example Z Preparation of7-(dimethylamino)-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]anilino]carbonyl)anilino}quinoliniumdichloride (Cpd. Z)

4-Chloro-7-dimethylaminoquinoline (Z1) [Konishi et al., WO 9611187 A1](70 mg, 0.34 mmol) was added to a solution of A7 (100 mg, 0.28 mmol) inEtOH (20 ml) and H₂O (10 mL) then c.HCl (0.25 mL, 9 eq) was added andrefluxed for 2 days. Mass spectrum of a sample of the reaction mixtureshowed A7 still present, so more 4-chloro-7-dimethylaminoquinoline (35mg, 0.17 mmol) was added and refluxed continued for a further 5 days.The reaction mixture was then evaporated to dryness. The residue wasdissolved in a small volume of MeOH diluted with EtOAc. The resultingprecipitate was filtered, and crystallized from MeOH/EtOAc to give crudeproduct (160 mg), which was purified by preparative HPLC (fraction withmass of 489⁺ve was evaporated to dryness, and the residue dissolved inEtOH/H₂O 2:1 (5 mL), diluted with EtOAc, and the resulting precipitatefiltered) to give Cpd. Z (20 mg 13%). This was 90% clean by HPLC; MP(MeOH EtOAc) 285-289° C. (dec); ¹H NMR [(CD₃)₂SO] δ 13.47 (br, 1H, N⁺H)10.54 (s, 1H, NH), 10.51 (s, 1H, NH), 10.49 (s, 1H, NH), 8.44 (d, J=9.7Hz, 1H, ArH), 8.35-8.30 (m, 3H, ArH) 8.13 (d, J=8.6 Hz, 2H, ArH), 7.98(t, J=1.9 Hz, 1H, ArH), 7.63-7.58 (m, 3H, ArH), 7.49 (t, J=8.1 Hz, 1H,ArH), 7.40 (dd, J=9.6, 2.5 Hz, 1H, ArH), 7.18 (d, J=7.5 Hz, 2H, ArH),7.09 (dd, J=7.7, 1.3 Hz, 1H, ArH), 6.81 (d, J=2.5 Hz, 1H, ArH), 6.71 (d,J=7.0 Hz, 1H, ArH), 3.98 (s, 3H, N⁺CH₃), 3.16 (s, 6H, NMe₂); MassAPCI⁺ve 489.

Example AA Preparation of4-[4-({4-[(7-dimethylamino)quinolin-4-ylamino)benzamido)-phenylamino)-1-methylpyridiniumdichloride (Cpd. AA)

To a solution of amine B7 (311 mg, 0.79 mmol) dissolved in 1:2 EtOH:H₂O(3 mL) were sequentially added c.HCl (0.22 mL, 0.72 mmol) and4-chloro-7-dimethylaminoquinoline (Z1) (175 mg, 0.85 mmol). Theresulting mixture was refluxed for 30 h (reaction progress followed byTLC, eluting with the top phase of a 5:4:1 mixture of n-BuOH:H₂O:AcOH;R_(f)=0.30-0.40, bright yellow spot at 365 nm). After this time, solventwas removed under reduced pressure. The residue was re-dissolved inMeOH, and solvent was again removed under reduced pressure. This latterprocess was repeated once more, and the residue was crystallized thricefrom MeOH:EtOAc to afford Cpd. AA as an amorphous yellow solid (93 mg,21%). ¹H NMR [CD₃)₂SO]: 3.15 [s, 6H, ArN(CH₃)₂], 3.96 [s, 3H, ArN⁺—CH₃],6.72 [d, J=7.00 Hz, 1H, ArH], 6.91 [d, J=2.36 Hz, 1H ArH], 7.14 [d,J=7.40 Hz, 2H, ArH], 7.36 [m, 3H, ArH], 7.62 [d, J=8.53 Hz, 2H, ArH],7.95 [d, J=8.89 Hz, 2H, ArH], 8.16 [d, J=8.53 Hz, 2H, ArH], 8.26 [d,J=7.40 Hz, 2H, ArH], 8.32 [d, J=7.00 Hz, 1H, ArH], 8.57 [d, J=9.59 Hz,1H, ArH], 10.53 [s, 1H, —NH—], 10.70 [s, 1H, —NH—], 10.75 [s, 1H, —NH—],13.85 [br s, 1H, Ar—N⁺H]. LCMS (APCI⁺): 490 (100%), 491 (20%). HPLC:96.7%.

Example BB Preparation ofN-[4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(7-(dimethylamino)quinolin-4-ylamino)benzamidehydrochloride (Cpd. BB)

To a solution of amide ES (306 mg, 0.83 mmol) in 1:2 EtOH:H₂O (10 mL)were sequentially added c.HCl (0.23 mL) and4-chloro-7-dimethylamino-quinoline (Z1) (188 mg, 0.91 mmol), and theresulting mixture was refluxed for 20 h. After this time [when MS andTLC analysis (5:4:1 n-BuOH:H₂O:CH₃CO₂H) of the reaction mixturesuggested the remaining small quantity of E5 was degrading], thereaction mixture was filtered, and the resulting solid washedsequentially with EtOAc and hexanes, to furnish Cpd. BB as an amorphouslemon-yellow solid (199 mg, 45%). ¹H NMR [CD₃)₂SO]: 2.28 [s, 3H, ArCH₃],3.15 [s, 6H, ArN(CH₃)₂], 6.18 [br s, 1H, ArH], 6.70 [d, J=7.02 Hz, 1H,ArH], 6.88 [d, J=2.46 Hz, 1H, ArH], 7.38 [dd, J=9.62, 2.46 Hz, 1H, ArH],7.61 [d, J=8.60, 2H, ArH], 7.81 [m, 5H, ArH & ArNH₂], 8.14 [d, J=8.60Hz, 2H, ArH], 8.31 [d, J=7.02 Hz, 1H, ArH], 8.55 [d, J=9.71 Hz, 1H,ArH], 10.41 [br s, 1H, ArH], 10.65 [m, 2H, ArNHAr & ArNHAr], 12.68 [v brs, 1H, ArC(O)NH], 13.77 [v br s, 1H, quinolinyl N⁺H]. LCMS (APCI⁺): 506(100%). HPLC: 96.6%.

Example CC Preparation of7-(dimethylamino)-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)aniline]quinoliniumdichloride (Cpd. CC)

4-Chloro-7-dimethylaminoquinoline (Z1) (71 mg, 0.34 mmol) and c.HCl (0.3mL, 9 eq) were sequentially added to a solution of C6 (104 mg, 0.0.29mmol) in EtOH (10 ml) and H₂O (5 mL), and the mixture was refluxed for 4days. The reaction mixture was then diluted with EtOAc (150 mL),refluxed, and then cooled to room temperature. The resulting precipitatewas filtered, and then dissolved in a small volume of MeOH. The solutionwas diluted with EtOAc, and the resulting precipitate filtered, and thenpurified by preparative HPLC to give Cpd. CC (13 mg, 8%): mp(MeOH/EtOAc) 188° C. (dec); ¹H NMR [(CD₃)₂SO] δ 13.22 (bd, J=5.7 Hz, 1H,N⁺H), 10.61 (s, 1H, NH), 10.51 (s, 1H, NH), 10.38 (s, 1H, NH), 8.42 (d,J=9.7 Hz, 1H, ArH), 8.32 (d, J=7.5 Hz, 2H, ArH), 8.23 (t, J=6.7 Hz, 1H,ArH), 7.96-7.92 (m, 3H, ArH), 7.88 (t, J=1.8 Hz, 1H, ArH), 7.69 (t,J=7.9 Hz, 1H, ArH), 7.58 (dd, J=8.0, 1.4 Hz, 1H, ArH), 7.43 (d, J=8.9Hz, 2H, ArH), 7.34 (dd, J=9.6, 2.6 Hz, 1H, ArH), 7.21 (d, J=7.5. Hz, 2H,ArH), 6.77 (d, J=2.5 Hz, 1H, ArH), 6.48 (d, J=7.1 Hz, 1H, ArH), 3.99 (s,3H, N⁺CH₃), 3.15 [s, 6H, N(CH₃)₂]. Mass APCI⁺ve 489.

Example DD Preparation of7-(dimethylamino)-4-[4-({4-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)aniline]quinoliniumdichloride (Cpd. DD)

4-Chloro-7-dimethylaminoquinoline (Z1) (140 mg, 0.62 mmol) and c.HCl(0.3 mL, 9 eq) were sequentially added to a solution of D5 (110 mg, 0.31mmol) in EtOH (20 ml) and H₂O (10 mL), and the mixture was refluxed for4 d. The reaction mixture was diluted with EtOAc (150 mL), refluxed, andthen cooled to room temperature. The resulting precipitate was filtered,and then dissolved in a small volume of MeOH, which was then dilutedwith EtOAc. The resulting precipitate was filtered to give crudeproduct, which was purified by preparative HPLC to give Cpd. DD (47 mg,27%) HPLC 99.9%; mp (MeOH/EtOAc) 198-200° C.; ¹H NMR [(CD₃)₂SO] δ 13.26(br, 1H, N⁺H), 10.71 (s, 1H, NH), 10.46 (s, 1H, NH), 10.37 (brs, 1H,NH), 8.43 (d, J=9.6 Hz, 1H, ArH), 8.37 (d, J=7.4 Hz, 2H, ArH), 8.23 (d,J=7.0 Hz, 1H, ArH), 8.11 (d, J=8.6 Hz, 2H, ArH), 7.96 (d, J=8.8 Hz, 2H,ArH), 7.50 (d, J=8.6 Hz, 2H, ArH), 7.43 (d, J=8.8 Hz, 2H, ArH), 7.34(dd, J=9.6, 2.4 Hz, 1H, ArH), 7.28 (d, J=7.5 Hz, 2H, ArH), 6.78 (d,J=2.4 Hz, 1H, ArH), 6.49 (d, J=7.0 Hz, 1H, ArH), 4.01 (s, 3H, N⁺CH₃),3.14 [s, 6H, N(CH₃)₂]. Mass APCI⁺ve 489.

TABLE 2 EXEMPLARY COMPOUNDS OF THE INVENTION (III)

For the entries in Table 2, R⁴ is H, A is NH, and X is H. Unlessspecified, R⁶-R⁹ are H. Unless specified, G7, G8, D1, and D2 are CH.HPLC Cpd. R⁶-R⁹ Y attach Z mp Formula (%) Anal A — CONH m Q1 253-257C₂₈H₂₇N₅OCl₂ 99.7 C, H, N V R⁸ = NH₂ CONH m Q1 281-285 C₂₈H₂₆N₆OCl₂ 99.9C, H, N K R⁷ = NH₂ CONH m Q1 270-274 C₂₈H₂₆N₆OCl₂ 97.9 C, H, N L R⁷ =NH₂ CONH p Q1 262-266 C₂₈H₂₆N₆OCl₂ 99.3 LRMS O R⁷ = NMe₂ CONH p Q1242-246 C₃₀H₃₀N₆OCl₂ 96.4 LRMS N R⁷ = NH₂ NHCO p Q1 >300 C₂₈H₂₆N₆OCl₂97.6 LRMS F R⁷ = NO₂ CONH m Q1 273-277 C₂₈H₂₄Cl₂N₆O₃ 97.0 HRMS R R⁸ =NO₂ CONH m Q1 303(dec.) C₂₈H₂₄Cl₂N₆O₃ 96.0 HRMS Y R⁸ = NH₂ NHCO p Q1290-295 C₂₈H₂₆N₆Ocl₂ 97.0 HRMS S R⁸ = NO₂ CONH p Q1 265(dec.)C₂₈H₂₄Cl₂N₆O₃ 98.1 LRMS AA R⁸ = NMe₂ CONH p Q1 242-245 C₃₀H₃₀Cl₂N₆O 97.6LRMS J R⁷ = NO₂ NHCO m Q1 >300 C₂₈H₂₄N₆Cl₂O₃ 98.5 HRMS T R⁸ = NO₂ NHCO mQ1 >300 C₂₈H₂₄N₆Cl₂O₃ 97.7 HRMS D — NHCO p Q1 >300 C₂₈H₂₅NCl₂N₅O 96.6HRMS C — NHCO m Q1 >300 C₂₈H₂₅NCl₂N₅O 97.5 HRMS U R⁸ = NO₂ NHCO p Q1 268dec C₂₈H₂₄N₆Cl₂O₃ 96.6 LRMS M R⁷ = NH₂ NHCO m Q1 >310 C₂₈H₂₆N₆OCl₂ 96.0HRMS X R⁸ = NH₂ NHCO m Q1 >300 C₂₈H₂₆N₆OCl₂ 95.8 HRMS BB R⁸ = NMe₂ CONHp Q2 272-275 C₂₉H₂₉ClN₈O 96.6 HRMS EEE7 R⁷ = NMe₂ CONH p Q2 260-265C₂₉H₂₉ClN₈O 97.6 LRMS H R⁷ = NO₂ CONH p Q2 301-305 C₂₇H₂₃ClN₈O₃ 98.5LRMS Q R⁷ = NMe₂ CONH m Q1 >300 C₃₀H₃₀Cl₂N₆O 97.8 LRMS Z R⁸ = NMe₂ CONHm Q1 285-289 C₃₀H₃₀Cl₂N₆O 90.0 HRMS B — CONH p Q1 >300 C₂₈H₂₅Cl₂N₅O 99.7LRMS DD R⁸ = NMe₂ NHCO p Q1 198-200 C₃₀H₃₀Cl₂N₆O 99.9 HRMS CC R⁸ = NMe₂NHCO m Q1 188 dec C₃₀H₃₀Cl₂N₆O 98.5 HRMS E — CONH p Q2 189-192C₂₇H₂₄ClN₇O 95.7 LRMS I R⁷ = NO₂ CONH p Q3 >300 C₂₅H₂₃ClN₈O 97.1 LRMS GR⁷ = NO₂ CONH p Q1 183-187 C₂₈H₂₄Cl₂N₆O₃ 99.9 LRMS EE R⁷ = NMe₂ NHCO mQ2 >300 C₂₉H₂₉ClN₈O 96.0 HRMS W R⁸ = NH₂ CONH p Q1 242-245 C₂₈H₂₆Cl₂N₆O96.1 LRMS OO2 R⁷ = NMe₂ NHCO p Q1 156 C₃₀H₃₀Cl₂N₆O 97.0 HRMS NN R⁷ =NMe₂ CONH p Q3 >280 C₂₇H₃₀Cl₂N₈O 98.7 LRMS OO1 R⁷ = NMe₂ NHCO m Q1170-173 C₃₀H₃₀Cl₂N₆O 98.0 HRMS QQ R⁷ = NMe₂ NHCO m Q3 >280 C₂₇H₃₀Cl₂N₈O96.0 HRMS EEE2 — CONH p Q9 171-174 C₂₇H₂₃Cl₂N₅O 100.0 LRMS PP R⁷ = NMe₂NHCO p Q3 >300 C₂₇H₃₀Cl₂N₈O 97.9 HRMS GG1 — NHCO p Q2 >300 C₂₇H₂₅Cl₂N₇O97.7 HRMS GG2 R⁸ = NH₂ NHCO p Q2 257-261 C₂₇H₂₆Cl₂N₈O 97.8 HRMS JJ1 R⁷ =NMe₂ CONH m Q3 278-282 C₂₇H₃₀Cl₂N₈O 95.8 LRMS JJ2 — NHCO m Q3 264-268C₂₅H₂₅Cl₂N₇O 96.4 LRMS GG3 R⁷ = NH₂ NHCO p Q2 >300 C₂₇H₂₆Cl₂N₈O 96.4HRMS II — CONH m Q2 251-255 C₂₇H₂₅Cl₂N₇O 95.4 LRMS FF1 — NHCO m Q2 >300C₂₇H₂₅Cl₂N₇O 99.5 HRMS FF2 — NHCO m Q4 >300 C₂₆H₂₄Cl₂N₈O 99.2 HRMS LL R⁷= Cl CONH p Q2 250-254 C₂₇H₂₄Cl₃N₇O 99.7 LRMS KK R⁷, R⁸, R⁹ = F CONH pQ2 >280 C₂₇H₂₂Cl₂F₃N₇O 98.9 LRMS HH R⁷ = NMe₂ NHCO p Q2 >300C₂₉H₃₀Cl₂N₈O 96.7 HRMS GG4 R⁷ = NO₂ NHCO p Q2 295-300 C₂₇H₂₄Cl₂N₈O₃ 96.4HRMS GG5 R⁸ = NO₂ NHCO p Q2 >300 C₂₇H₂₄Cl₂N₈O₃ 96.6 HRMS RR1 R⁷ = NO₂NHCO m Q4 >300 C₂₆H₂₃Cl₂N₉O₃ 96.0 HRMS RR2 R⁷ = NO₂ NHCO m Q2 >300C₂₇H₂₄Cl₂N₈O₃ 99.0 HRMS RR3 R⁷ = NH₂ NHCO m Q2 280-284 C₂₇H₂₆Cl₂N₈O 98.0LRMS N1 R⁷ = NO₂ NHCO p Q1 >300 C₂₈H₂₄Cl₂N₆O₃ 97.0 LRMS SS R⁷ = NMe₂NHCO p Q4 283-287 C₂₈H₂₉Cl₂N₉O 99.2 LRMS TT — NHCO p Q4 263-267C₂₆H₂₄Cl₂N₈O 98.3 LRMS UU R⁷ = NH₂ NHCO m Q4 250-255 C₂₆H₂₅Cl₂N₉O 99.5HRMS VV1 R⁷ = NO₂ NHCO p Q4 260-265 C₂₆H₂₃Cl₂N₉O₃ 100.0 HRMS VV2 R⁷ =NH₂ NHCO p Q4 262-266 C₂₆H₂₅Cl₂N₉O 99.7 HRMS WW — CONMe p Q9 287-292C₂₈H₂₅Cl₂N₅O 96.3 HRMS XX* — NHCO p Q2 >290 C₂₆H₂₄Cl₂N₈O 99.2 C, H, NYY** — NHCO p Q2 >290 C₂₆H₂₄Cl₂N₈O 98.9 C, H, N ZZ*** — NHCO p Q2 >295C₂₆H₂₄Cl₂N₈O 100.0 C, H, N *G₈ = N; **D₂ = N; ***D₁ = N

Example EE Synthesis of4-[4-({3-[(2-amino-6-methyl-4-pyrimidinyl)amino]benzoyl}amino)anilino]-6-(dimethylamino)quinoliniumchloride (Cpd. EE)

4-[4-(acetylamino)anilino]-6-nitroquinolinium chloride (A3). To asolution of N-(4-aminophenyl)acetamide (A1) (933 mg, 6.2 mmol) inethanol (30 mL) was added 6-nitro-4-chloroquinoline (A2) (1.08 g, 5.18mmol) in ethanol (10 mL) followed by a solution of 4N HCl in 1,4-dioxane(1.5 mL). The reaction mixture was refluxed for 30 min, by this time TLC(SiO₂/8% MeOH/DCM) showed completion of the reaction. The reactionmixture was diluted with EtOAc and brought to boil, then allowed to coolto 20° C. The resulting precipitate was filtered, washed with EtOAc andrecrytallized from MeOH to give A3 (1.79 g, 96%); mp (MeOH)>280° C.; ¹HNMR [(CD₃)₂SO] δ 14.59 (br, 1H, N⁺H), 11.36 (br, 1H, NH), 10.24 (s, 1H,NH), 9.78 (d, J=2.3 Hz, 1H, ArH), 8.71 (dd, J=9.3, 2.3 Hz, 1H, ArH),8.57 (d, J=7.0 Hz, 1H, ArH), 8.21 (d, J=9.3 Hz, 1H, ArH), 7.80 (d, J=8.8Hz, 2H, ArH), 7.42 (d, J=8.8 Hz, 2H, ArH), 6.86 (d, J=7.0 Hz, 1H, ArH),2.09 (s, 3H, COCH₃), APCI⁺ve 323.

4-[4-(acetylamino)anilino]-6-aminoquinolinium chloride (A4). To asuspension of (A3 (1.75 g, 4.88 mmol) in MeOH (10 mL) was added 10% Pd/C(0.1 g) and hydrogenated (under 40 psi), for 2 h. The reaction mixturewas filtered and washed with more MeOH. The filtrated was concentratedto half the volume, diluted with EtOAc and the rest of the MeOH wasevaporated. The Resulting precipitate was filtered washed with moreEtOAc and dried to give essentially pure (A4) (1.255 g 78%); mp(MeOH/EtOAc)>280° C.; ¹H NMR [(CD₃)₂SO] δ 13.95 (br, 1H, N⁺H), 10.14 (s,1H, NH), 10.01 (s, 1 H, NH), 8.10 (d, J=6.7 Hz, 1H, ArH), 7.74 (d, J=8.9Hz, 3H, ArH), 7.43 (d, J=2.2 Hz, 1H, ArH), 7.38-7.32 (m, 3H, ArH), 6.61(d, J=6.7 Hz, 1H, ArH), 5.94 (s, 2H, NH₂), 2.08 (s, 3H, COCH₃); APCI+ve293.

N-(4-{[6-(Dimethylamino)-4-quinolinyl]amino}phenyl)acetamide (A5). To asolution of A4 (1.237 g, 3.76 mmol), in MeOH (50 mL) was added 38%aqueous formaldehyde (8.4 mL, 112.8 mmol, 30 eq), NaBH3CN (2.134 g,30.08 mmol, 8 eq), NaOAc (526 mg, 6.41 mmol) and 2 drops of c. HCl. Thereaction mixture was stirred at 20° C. for 2 h, by this time TLC(SiO₂/8% MeOH/DCM/aq NH3) and mass spectrum showed completion of thereaction. Then the reaction mixture was acidified with c. HCl, stirredfor 1 h. at 20° C. After this time the reaction mixture was carefullyneutralize with aqueous NH₃. The MeOH was evaporated under reducedpressure and the aqueous residue was the stirred with aqueous NH₃. Theresulting precipitate was filtered washed with water to give essentiallypure A5 (1.22 g, 91%); This was used without further purification; mp,(MeOH)>280° C.; ¹H NMR [(CD₃)₂SO] δ 9.95 (s, 1H, NH), 8.85 (bs, 1H, NH),8.18 (d, J=5.5 Hz, 1H, ArH), 7.72 (d, J=9.3 Hz, 1H, ArH), 7.65 (d, J=8.8Hz, 2H, ArH), 7.41 (dd, J=9.3, 2.6 Hz, 1H, ArH), 7.29-7.26 (m, 3H, ArH),6.67 (d, J=5.5 Hz, 1H, ArH), 3.06 [s, 6H, N(CH₃)₂], 2.06 (s, 3H, COCH₃);APCI⁺ve 321.

4-(4-aminoanilino)-6-(dimethylamino)quinolinium chloride (A6). To asuspension of A5 in 1,4-dioxane (20 mL) was added 1.5 M aq HCl (5 mL)and the reaction mixture was refluxed for 2 h. The reaction mixture wasthen diluted with MeOH/EtOAc, brought to boil and allowed to cool to 20°C. The resulting precipitate was filtered, washed with EtOAc and driedto give essentially pure A6 as a pale yellow solid (1.20 g, 100%); mp(MeOH/EtOAc) 152-155; ¹H NMR [(CD₃)₂SO] 14.35 (br, 1H, N⁺H), 10.50 (br,2H, NH₂), 8.27 (d, J=6.6 Hz, 1H, ArH), 7.92 (d, J=9.4 Hz, 1H, ArH), 7.65(dd, J=9.5, 2.6 Hz, 1H, ArH), 7.57 (d, J=2.4 Hz, 1H, ArH), 7.54 (d,J=8.5 Hz, 2H, ArH), 7.41 (d, J=8.1 Hz, 2 H, ArH), 6.68 (d, J=6.7 Hz, 1H,ArH), 3.12 (s, 6H, [N(CH₃)₂]; APCI⁺ve 279.

3-[(2-Amino-6-methyl-4-pyrimidinyl)amino]benzoic acid hydrochloride(A9). 3-Amino benzoic acid (A7) (1.02 g, 7.42 mmol) and2-amino-6-chloro-4-methylpyrimidine (A8) (1.08 g, 7.42 mmol) wasdissolved in 2-ethoxyethanol by heating, then a drop of c.HCl was added,refluxed for 1 h. and cool to 20° C. The resulting precipitate wasfiltered washed with more 2-ethoxy ethanol followed by EtOAc. The solidwas recrystallized from MeOH/EtOAc give A9 (1.98 g, 95%) as an off whitesolid; mp>300° C.; ¹H NMR [(CD₃)₂SO] δ 12.97 (bs 2H, NH, and COOH),10.81 (s, 1H, NH), 8.22 (bs, 1H, ArH), 8.03 (s, 1H, ArH), 7.82 (br, 2H,NH₂), 7.72 (bd, J=7.7 Hz, 1H, ArH), 7.50 (t, J=7.9 Hz, 1H, ArH), 6.22(s, 1H, ArH); APCI⁺ ve 245.

4-[4-({3-[(2-amino-6-methyl-4-pyrimidinyl)amino]benzoyl}-amino)anilino]-6-(dimethylamino)quinoliniumchloride (15) (Cpd. EE). A mixture of4-(4-aminoanilino)-6-(dimethylamino)quinolinium chloride (A6) (102 mg,0.32 mmol), A9 (111 mg, 0.32 mmol), and EDCI (151 mg, 0.64 mmol) in DMF(5 mL) was stirred at 20° C. for 5 min. Then DMAP (96 mg, 0.64 mmol) wasadded and the reaction mixture was stirred at 20° C. for 72 h. Thesolvent was removed under reduced pressure and residue was diluted withH₂O and basified with aqueous NH₃. The resulting precipitate wasfiltered washed with water, and purified by chromatography in SiO,eluting with a gradient 0-5% DCM/MeOH and 1% aqueous NH₃ to give 102 mg62%. A sample of this (98 mg, 0.19 mmol) was dissolved in MeOH (5 mL)and 4N HCl in 1,4-dioxane (0.3 mL) was added and stirred for 30 min.This was then diluted with EtOAc and the resulting precipitate wasfiltered and recrystallized from MeOH/EtOAc to give Cpd. EE (107 mg); ¹HNMR [(CD₃)₂SO] δ (13.9 br 1H, N⁺H), 12.9 (br (1 H, N⁺H), 10.58 (s, 2H,2×NH), 10.40 (s, 1H, NH), 8.26 (d, J=6.8 Hz, 1H, ArH), 8.38 (br 1H,ArH), 8.06 (bs, 1H, ArH), 8.00 (d, J=8.9 Hz, 2H, ArH), 7.89 (d, J=9.4Hz, 1H, ArH), 7.75 (bd, J=7.6 Hz, 1H, ArH), 7.56-7.52 (m, 2H, ArH), 7.46(d, J=8.8 Hz, 2H, ArH), 6.67 (d, J=6.8 Hz, 1H), 6.19 (s, 1 H, ArH), 3.12[s, 6H, (NCH₃)₂], 2.28 (s, 3H, CH₃), The signal for NH₂ was notobserved, APCI⁺ve 505.

Example FF Preparation of3-[(2-amino-6-methyl-4-pyrimidinyl)amino]-N-[4-(4-quinolinylamino)phenyl]benzamidedihydrochloride (Cpd. FF1) and related compound (Cpd. FF2)

3-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-(4-nitrophenyl)-benzamidehydrochloride (B2). 3-Amino-N-(4-nitrophenyl)benzamide (B1) (1.0 g, 3.89mmol) and 4-chloro-6-methyl-2-pyrimidinylamine (A8) (569 mg, 3.89 mmol)were dissolved in warm 2-ethoxyethanol. (20 mL). Two drops c.HCl wasthen added to the reaction mixture and refluxed for 1 h. by this timeTLC and mass spec. showed completion of the reaction. The reactionmixture was diluted with ethyl acetate and cooled to 20° C. Theresulting precipitate was filtered, washed with more EtOAc andrecrystallized from MeOH/EtOAc/Charcoal/Celite to give B2 (1.531 g,98%); m.p (MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 12.86 (br s, 1H,N⁺H), 10.90 (s, 1H, NH), 10.80 (br s, 1H, NH), 8.30-8.26 (m, 2 H, ArH),8.11-8.08 (m, 4H, ArH), 7.80-7.78 (br m, 3H, ArH & NH₂), 7.56 (t, J=7.8Hz, 1H, ArH), 2.30 (s, 3H, CH₃); mass APCI⁺ 365.

3-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-(4-aminophenyl)-benzamidehydrochloride (B3). To a suspension of compound B2 (1.41 g, 3.51 mmol)in (MeOH) 30 (mL) was added 10% Pd/C (20 mg) and hydrogenated under 45Hg mm for 5 h. The reaction mixture was filtered through a pad of celiteand the filtrate was evaporated to dryness. The residue was dissolved ina small volume of MeOH then 1 ml of 10.25M HCl in MeOH was added,stirred 10 min and evaporated to dryness. The residue was recrystallizedfrom MeOH/EtOAc to give B3 (758 mg, 58%); m.p. (MeOH/EtOAc); ¹H NMR[(CD₃)₂SO] δ 12.70 (br, 1H, NH), 10.83 (br s, 1H, NH), 10.43 (s, 1H,NH), 9.70 (v br, 2H, NH₂), 8.05-7.84 (br m, 2H, ArH), 7.85-7.75 (m, 4H,ArH & NH₂), 7.55 (t, J=7.9 Hz, 1H, ArH), 7.28 (d, J=8.7 Hz, 2H, ArH),6.24 (s, 1H, ArH), 2.30 (s, 3H, CH₃); mass APCI⁺ 335.

3-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-[4-(4-quinolinyl-amino)phenyl]benzamidedihydrochloride (Cpd. FF1). To a solution of compound B3 (150 mg, 0.37mmol) in EtOH (15 mL) and H₂O (7.5 mL) was added 4-chloroquinoline (73mg, 0.45 mmol) and stirred until it dissolved, then 2 drops of c. HClwas added. The reaction mixture was refluxed for 20 h, diluted withEtOAc, brought to boil and cool to 20° C. The resulting precipitate wasfiltered and recrystallized from MeOH/EtOAc/Charcoal/Celite to give Cpd.FF1 (195 mg, 96%) as a yellow solid.; M.P (MeOH/EtOAc)>300° C.; ¹H NMR[(CD₃)₂SO] δ 13.72 (v br, 2H, 2×N⁺H), 10.91 (br s, 1H, NH), 10.96 (br s,1H, NH), 10.56 (s, 1H, HN), 8.79 (d, J=8.5 Hz, 1H, ArH), 7.50 (d, J=6.9Hz, 1H, ArH), 8.14-7.99 (m, 6H, ArH & NH₂), 7.83 (m, 3H, 7.55 (t, J=7.9Hz, 1H, ArH), 7.49 (d, J=8.8 Hz, 2H, ArH), 6.78 (d, J=6.9 Hz, 1H, ArH),6.24 (s, 1H, ArH), 2.30 (s, 3H, ArH), mass APCI⁺ 476.

3-[(2,6-Diamino-4-pyrimidinyl)amino]-N-(4-nitrophenyl)-benzamidehydrochloride (B5). 3-Amino-N-(4-nitrophenyl)benzamide (B1) (1.07 g,4.14 mmol) and 4-chloro-2,6-diaminopyrimidine (B4) (569 mg, 3.89 mmol)were dissolved in warm 2-ethoxyethanol (20 mL). Two drops c. HCl wasthen added to the reaction mixture and refluxed for 20 h., by this timeTLC and mass spec showed the completion of the reaction. The reactionmixture was diluted with ethyl acetate and cooled to 20° C. Theresulting precipitate was filtered, washed with more EtOAc andrecrystallized from MeOH/EtOAc/Charcoal/Celite to give compound B5 (824mg, 50%), m.p (MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 11.57 (br, 1H,N⁺H), 10.84 (s, 1H, NH), 9.98 (s, 1H, NH), 8.30-8.26 (m, 2H, Ar),8.10-8.07 (m, 2H, ArH), 8.06 (br s, 2H, NH₂), 7.70 (d, J=7.2 Hz, 1H,ArH), 7.53-7.49 (m, 3H, ArH), 7.38 (br s, 2H, NH₂), mass APCI⁺ 366.

N-(4-Aminophenyl)-3-[(2,6-diamino-4-pyrimidinyl)amino]-benzamidehydrochloride (B6). To a suspension of compound B5 (567 mg, 1.41 mmol)in (MeOH) (30 mL) was added 10% Pd/C (20 mg) and hydrogenated under 40Hg mm for 6 h. The reaction mixture was filtered through a pad of celiteand the filtrate was evaporated to dryness. The residue was dissolved ina small volume of MeOH then 1 ml of 1.25M HCl in MeOH was added, stirred10 min and evaporated to dryness. The residue was recrystallized fromMeOH/EtOAc to give B6 (398 mg, 76%); mp (MeOH/EtOAc)>300° C., ¹H NMR[(CD₃)₂SO] δ 10.37 (s, 1H, NH), 9.93 (s, 1H, NH), 7.92-7.80 (m, 4H, ArH& NH₂), 7.68 (d, J=7.7 Hz, 1H, ArH), 7.61 (br s, 2H, NH₂), 7.49-7.45 (m,3H, ArH), 7.24 (br d, J=8.7 Hz, 2H, ArH), 5.45 (s, 1H, ArH), mass APCI⁺336.

3-[(2,6-Diamino-4-pyrimidinyl)amino]-N-[4-(4-quinolinylamino)-phenyl]benzamidedihydrochloride (Cpd. FF2). To a solution of compound B6 (150 mg, 0.37mmol) in EtOH (15 mL) and H₂O (7.5 mL) was added 4-chloroquinoline (73mg, 0.45 mmol) and stirred until it dissolved, then 2 drops of c.HCl wasadded. The reaction mixture was refluxed for 20 h, diluted with EtOAc,brought to boil and cool to 20° C. The resulting precipitate wasfiltered and stirred in aqueous NH₃ (20 ml) to convert to free base.This new precipitate was filtered and chromatographed (neutral Al₂O₃,0-7% DCM/MeOH) to give pure free base of the product. This was thenconverted to HCl salt by using 1.25 M HCl in MeOH to give Cpd. FF2 (139mg, 70%); m.p (MeOH/EtOAc)>300° C. ¹H NMR [(CD₃)₂SO] δ 14.25 (br, 1H,N⁺H), 12.00 (br, 1H, N⁺H), 10.95 (s, 1H, NH), 10.53 (s, 1H, NH), 9.93(s, 1H, NH), 9.05 (d, J=8.5 Hz, 1H, ArH), 7.51 (d, J=7.0 Hz, 1H, ArH),8.06-8.00 (m, 5H, ArH), 7.95-7.91 (br m, 2H, NH₂), 7.81 (d d, J=7.6, 1.4Hz, 1H, ArH), 7.71 (d, J=7.6 Hz, 1H, ArH), 7.58 (s, 2H, NH₂), 7.57-7.47(m, 5H, ArH), 6.78 (d, J=7.0 Hz, 1H, ArH), 6.78 (d, J=7.0 Hz, 1H, ArH),5.46 (s, 1H, ArH); HRMS (FAB⁺) calc. for C₂₆H₂₃N₈O (M⁺¹) m/z 463.1995,found 463. 1992.

Example GG Preparation of4-[(2-amino-6-methyl-4-pyrimidinyl)amino]-N-[4-(4-quinolinylamino)phenyl]benzamidedihydrochloride (Cpd. GG1) and Related Compounds (Cpd. GG2, Cpd. GG3,Cpd. GG4, Cpd. GG5)

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-(4-nitrophenyl)-benzamide(5). 4-Amino-N-(4-nitrophenyl)benzamide (C1) (730 mg, 2.84 mmol) and4-chloro-6-methyl-2-pyrimidinylamine (A8) (416 mg, 2.84 mmol) weredissolved in warm 2-ethoxyethanol (20 mL), two drops c.HCl was thenadded to the reaction mixture was refluxed for 40 min. by which time TLCand MS showed the completion of the reaction. The reaction mixture wasdiluted with ethyl acetate and cooled to 20° C. The resultingprecipitate was filtered and washed with more EtOAc to give a solidproduct. This was suspended in MeON and basified with aqueous NH₃ anddiluted with water. The resulting precipitate was filtered and dried togive C2 (671 mg). The filtrate was concentrated to give a further 276 mgof C2 (92% overall): mp (MeOH)>300° C.; ¹H NMR [(CD₃)₂SO] δ 10.59 (s,1H, NH), 9.37 (s, 1H, NH), 8.27-8.24 (m, 2H, ArH), 8.09-8.06 (m, 2H,ArH), 8.05-7.90 (m, 4H, ArH), 6.25 (s, 2H, NH₂), 5.96 (s, 1H, ArH), 2.16(s, 3H, CH₃); Mass APCI⁺ 365.

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-(4-aminophenyl)-benzamide(C3). A suspension of C2 (671 mg, 1.84 mmol) in MeOH (50 mL) washydrogenated (10% Pd/C 50 mg) at 40 Hg mm. for 3 h. The product wasprecipitated as a white solid. The reaction mixture was stirred in(MeOH/HCl/H₂O) (100 mL/2 mL/50 mL) and filtered to remove Pd/C residues.The filtrate was evaporated to dryness and crystallized from MeOH/EtOActo give C3 (740 mg, 99%); M.P. (MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ10.96 (bs, 1H, NH), 10.38 (s, 1H NH), 8.02-7.97 (m, 4H, ArH), 7.86 (d,J=8.8 Hz, 2H, ArH), 7.30 (d, J=8.8 Hz, 2H, ArH), 6.30 (d, J=0.6 Hz, 1H,ArH), 2.33 (s, 3H, CH₃). The signal for 2×NH₂ groups were not observed;mass spectrum APCI⁺ 335.

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-[4-(4-quinolinyl-amino)phenyl]benzamidedihydrochloride (Cpd. GG1). To a solution of C3 (218 mg, 0.54 mmol) inEtOH (20 mL) and H₂O (10 mL) was added 4-chloroquinoline (96 mg, 0.59mmol) and stirred until it dissolved, then 2 drops of c. HCl was added.The reaction mixture was refluxed for 3 h, diluted with EtOAc, broughtto boil and cool to 20° C. The resulting precipitate was filtered andrecrystallized from MeOH/EtOAc/Charcoal/Celite to give Cpd. GG1 (259 mg98%) as a yellow solid.; M.P (MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ13.75 (br. 2H, 2×N⁺H), 11.07 (s, 1H, NH), 10.99 (s, 1H, NH), 10.52 (s,1H, NH), 8.83 (d, J=8.4 Hz, 1H, ArH), 8.50 (d, J=7.0 Hz, 1H, ArH),8.11-7.99 (m, 10H, ArH & NH₂), 7.80 (dt, J=8.0, 1.1 Hz, 1H, ArH), 7.48(d, J=8.9 Hz, 2H, ArH), 6.78 (d, J=7.0 Hz, 1H, ArH), 6.35 (s, 1H, ArH),2.31 (s, 3H, CH₃); ¹³C NMR [(CD₃)₂SO] δ 164.8, 161.6, 155.9, 155.0,153.3, 142.5, 141.4, 138.4, 138.1, 133.7, 132.1, 129.6, 128.5 (2×C),126.8, 125.8 (2×C), 123.5, 121.4 (2×C), 120.2, 120.1, 116.9, 99.6, 94.3,18.4, one of the quaternary carbons were difficult to observe; HRMS FAB⁺calc. for C₂₇H₂₅N₇O (M⁺¹) m/z, 462.2042 found 462.2047.

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-{4-[(6-nitro-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. GG4). To a solution of compound C3 (200 mg, 0.49mmol) in EtOH (20 mL) and H₂O (10 mL) was added4-chloro-6-nitroquinoline (113 mg, 0.54 mmol) and stirred until itdissolved, then 2 drops of c. HCl was added. The reaction mixture wasrefluxed for 3 h., diluted with EtOAc, brought to boil and cool to 20°C. The resulting precipitate was filtered and recrystallized fromMeOH/EtOAc/Charcoal/Celite to give Cpd. GG4 (247 mg 87%) as a yellowsolid.; M.P (MeOH/EtOAc) 295-300° C.; ¹H NMR [(CD₃)₂SO] δ 13.50 (br, 2H,2×N⁺H), 11.32 (br, 1H, NH), 11.00 (brs, 1H, NH), 10.51 (s, 1H, NH), 9.81(d, J=2.2 Hz, 1H, ArH), 8.69 (dd, J=9.3, 2.2 Hz, 1H, ArH), 8.59 (d,J=6.9 Hz, 1H, ArH), 8.24 (d, J=9.3 Hz, 1H, ArH), 8.06-7.98 (m, 7H, ArH &NH₂), 7.48 (d, J=8.9 Hz, 2H, ArH), 6.90 (d, J=6.9 Hz, 1H, ArH), 6.32 (s,1H, ArH), 2.32 (d, J=0.4 Hz, 3H, CH₃); HRMS (FAB⁺) calc. for C₂₇H₂₃N₈O₃(M⁺¹) m/z 507.1893, found 507.1896.

4-[(2-amino-6-methyl-4-pyrimidinyl)amino]-N-{4-[(6-amino-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. GG3). To a suspension of Cpd. GG4 (140 mg, 0.24mmol) in (MeOH) 30 (mL) was added 10% Pd/C and hydrogenated under 45 Hgmm for 1 h. The reaction mixture was filtered through a pad of celiteand the filtrate was evaporated to dryness. The residue was dissolved ina small volume of MeOH then 1 ml of 1.25M HCl in MeOH was added, stirred10 min and evaporated to dryness. The residue was recrystallized fromMeOH/EtOAc to give Cpd. GG3 (107 mg, 81%); m.p. (MeOH/EtOAc)>300° C. ¹HNMR [(CD₃)₂SO] δ 14.13 (d, J=5.6 Hz, 1H, N⁺H), 12.95 (s, 1H, N⁺H), 10.97(s, 1H, NH), 10.44, (s, 1H, NH), 10.20 (s, 1H, NH), 8.22 (t, J=6.5 Hz,1H, ArH), 8.05-7.96 (m, 7H, ArH), 7.79 (d, J=9.1 Hz, 1H, ArH), 7.49 (d,J=1.9 Hz, 1H, ArH), 7.43-7.38 (m, 3H, ArH), 6.66 (d, J=6.8 Hz, ArH),6.30 (s, 1H, ArH), 2.24 (d, J=0.6 Hz, 3H, CH₃); HRMS (FAB⁺) calc. forC₂₇H₂₅N₈O (M⁺¹) m/z 477.2151, found 477.2153.

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-{4-[(7-nitro-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. GG5). To a solution of C3 (235 mg, 0.58 mmol) inEtOH (20 mL) and H₂O (10 mL) was added 4-chloro-7-nitroquinoline (132mg, 0.63 mmol) and stirred until it dissolved, then 2 drops of c.HCl wasadded. The reaction mixture was refluxed for 5 h., diluted with EtOAc,brought to boil and cool to 20° C. The resulting precipitate wasfiltered and recrystallized from MeOH/EtOAc/Charcoal/Celite to give Cpd.GG5 (317 mg 94%) as a yellow solid.; m.p (MeOH/EtOAc)>300° C.; ¹H NMR[(CD₃)₂SO] δ 12.9 (br, 1H, N⁺H), 10.81 (brs, 2 H, 2×NH), 10.44 (s, 1H,NH), 8.93 (d, J=9.3 Hz, 1H, ArH), 8.81 (d, J=1.9 Hz, 1H, ArH), 8.66 (d,J=6.6 Hz, 1H, ArH), 8.46 (d, J=8.9 Hz, 1H, ArH), 8.05-7.98 (m, 8H, ArH &NH₂), 7.47 (d, J=8.8 Hz, 1H, ArH), 6.94 (d, J=6.6 Hz, 1H, ArH), 6.26 (s,1H, ArH), 2.32 (s, 3H, CH₃); HRMS (FAB⁺) calc. for C₂₇H₂₃N₈O₃ (M⁺¹) m/z507.1893, found 507.1885.

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-{4-[(7-amino-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. GG2). To a suspension of Cpd. GG5 (150 mg, 0.25mmol) in (MeOH) 30 (mL) was added 10% Pd/C (20 mg) and hydrogenatedunder 45 Hg mm for 1 h. The reaction mixture was filtered through a padof celite and the filtrate was evaporated to dryness. The residue wasdissolved in a small volume of MeOH then 1 ml of 1.25M HCl in MeOH wasadded, stirred 10 min and evaporated to dryness. The residue wasrecrystallized from MeOH/EtOAc to give Cpd. GG2 (141 mg, 100%), m.p(MeOH/EtOAc) 257-262° C. ¹H NMR [(CD₃)₂SO] δ 13.45 (br, 2 H, 2×N⁺H),10.33 (s, 1H, NH), 10.30 (s, 1H, NH), 10.00 (br, 1H, NH), 8.33 (d, J=9.4Hz, 1H, ArH), 8.14 (d, J=7.0 Hz, 1H, ArH), 3.19-7.92 (m, 6H, ArH), 7.39(d, J=8.9 Hz, 2H, ArH), 7.04-7.01 (br m, 3H, ArH), 6.86 (d, J=2.2 Hz,1H, ArH), 6.67 (br s, 2H, NH₂), 6.43 (d, J=7.0 Hz, 1H, ArH &NH₂)), 6.13(s, 1H, ArH), 2.22 (s, 3H, CH₃); HRMS (FAB⁺) calc. for C₂₇H₂₅N₈O (M⁺¹)m/z 477.2151, found 477.2153.

Example HH Preparation of4-[(2-amino-6-methyl-4-pyrimidinyl)amino]-N-(4-{[6-(dimethylamino)-4-quinolinyl]amino}phenyl)benzamidedihydrochloride (Cpd. HH)

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]benzoic acid hydrochloride(D2). p-Aminobenzoic acid (D1) (1.48 g, 10.8 mmol) and4-chloro-6-methyl-2-pyrimidinamine (A8) (1.60 g, 11.9 mmol) wasdissolved in warm 2-ethoxyethanol (20 mL), then two drops of c. HCl wasadded and refluxed for 1 h. The reaction mixture was cooled to 20° C.and the resulting precipitate was filtered washed with more2-ethoxyethanol and EtOAc. The solid was boiled in MeOH, cooled andfiltered washed with more MeOH and dried to give D2 (2.89 g, 95%); m.p.(MeOH)>300° C.; ¹H NMR [(CD₃)₂SO] δ 12.93 (br, 1H, N⁺H or COOH), 12.80(br, 1H, N⁺H or COOH), 10.87 (s, 1H, NH), 7.92 (s, 6H, ArH & NH₂)), 6.27(s, 1H, ArH), 2.32 (s, 3H, CH₃); mass APCI⁺ 245.

4-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-(4-{[6-(dimethylamino)-4-quinolinyl]amino}phenyl)benzamidedihydrochloride (Cpd. HH). Compound D2 (111 mg, 0.4 mmol), EDCI (155 mg,0.72 mmol) and DMAP (174 mg, 1.44 mmol) in N-methylpyrrolidinone (5 mL)was stirred 5 min at 20° C.N⁴-(4-Aminophenyl)-N⁶,N⁶-dimethyl-4,6-quinolinediamine (100 mg, 0.36mmol) was then added and the reaction mixture was stirred at 20° C. for20 h. The reaction mixture was diluted with water and stirred. Theresulting precipitate was filtered washed with water and air dried. Thissolid was dissolved in hot MeOH, boiled with charcoal and filteredthrough a pad of celite. The filtrate was evaporated to dryness and theresulting residue was dissolved in MeOH (10 mL), 1.25 M HCl in MeOH (1mL) was added and stirred 10 min. The solvent was evaporated and theresidue was recrytallized from MeOH/EtOAc to give Cpd. HH (135 mg, 65%)as a yellow solid; m.p. (MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 14.10(br, 1H, N⁺H), 12.75 (br, 1H, N⁺H), 10.85 (br, 1H, NH), 10.45 (s, 1H,NH), 10.40 (s, 1H, NH), 8.27 (d, J=6.8 Hz, 1H, ArH), 8.08-7.99 (m, 6H,ArH), 7.88 (d, J=9.4 Hz, 1H, ArH), 7.65 (dd, J=9.4, 2.5 Hz, 1H, ArH),7.54 (d, J=2.4 Hz, 1H, ArH), 7.45 (d, J=8.9 Hz, 2H, ArH), 6.67 (d, J=6.8Hz, 1H, ArH), 6.27 (s, 1H, ArH), 3.12 [s, 6H, N(CH₃)₂], 2.32 (s, 3H,CH₃); mass APCI⁺ 505.

Example II Preparation ofN-[3-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. II)

6-Methyl-N⁴-(3-nitrophenyl)pyrimidine-2,4-diamine (E1). To a suspensionof 2-amino-4-chloro-6-methyl-pyrimidine (A8) [10.04 g, 69.93 mmol] and3-nitroaniline 99.95 g, 72.02 mmol) in 2-ethoxyethanol (300 mL) wasadded c.HCl (30 mL), and the resulting solution was refluxed for ˜16 h.After this time, the reaction mixture was allowed to cool to roomtemperature, and then diluted with brine and H₂O. The resultingsuspension was filtered through a Celite pad, and the solid materialthus collected re-dissolved in MeOH, and filtered through a pad ofCelite. Solvent was removed under reduced pressure to give diamine E1 asan amorphous beige solid, which was used without further purification:¹H NMR (400 MHz, DMSO): δ 12.97 (br s, 1H, pyrimidinyl-N⁺H), 11.03 (s,1H, ArNHAr), 8.52 (s, 1H, ArH), 8.31 (d, J=7.83 Hz, 1H, ArH), 7.97 (ddd,J=8.20, 2.19, 0.72 Hz, 1H, ArH), 7.83 (v br s, 2H, ArNH₂), 7.66 (t,J=8.21 Hz, 1H, ArH), 6.25 (s, 1H, ArH), 2.31 (s, 3H, ArCH₃); LCMS(APCI⁺): 246 (100%).

N⁴-(3-Aminophenyl)-6-methylpyrimidine-2,4-diamine (E2). To a refluxingsuspension of diamine E1 in 2:1 EtOH:H₂O (500 mL) were sequentiallyadded Fe dust (15.6 g, 4 molar equivalents wrt 1) and c.HCl (10 mL), andthe resulting mixture was refluxed overnight. After this time, the hotreaction mixture was filtered through a pad of Celite, and solvent wasremoved under reduced pressure. The residue was re-dissolved in hot H₂O,and the resulting suspension filtered through a pad of Celite. Solventwas removed under reduced pressure, and the residue dried via threeMeOH-azeotrope cycles. The resulting residue was reprecipitated fromacidified MeOH:EtOAc to give diamine E2 (5.36 g, 30% from A8) as anamorphous tan solid; ¹H NMR (400 MHz, DMSO): δ 12.90 (v v br s, 1H,pyrimidinyl-N⁺—H), 11.00 (br s, 1H, ArH), 9.75 (v v br s, 2H, ArNH₂),8.20-7.50 (m, 4H, ArH & ArNH₂), 7.37 (t, J=8.05 Hz, 1H, ArH), 6.98 (d,J=7.82 Hz, ArH, 1H), 6.26 (s, 1H, ArH), 2.29 (s, 3H, ArCH₃); LCMS(APCI⁺): 216 (100%), 217 (40%).

N-[3-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl]-4-nitrobenzamide (E3).To a suspension of diamine E2 (5.25 g, 20.85 mmol) and dry pyridine(8.40 mL, 104.26 mmol) in dry dioxane (200 mL) was added 4-nitrobenzoylchloride (10.79 g, 58.13 mmol), and the resulting solution was refluxedfor ˜14 h. After this time, the reaction mixture was cooled to roomtemperature, and then basified by addition of aqueous ammonia solution.The resulting solution was diluted with H₂O, and the resultingprecipitate collected by filtration to afford amide E3 as an amorphousyellow-brown solid (7.89 g, 94%) [a small sample of Phis crude materialwas reprecipitated from acidified MeOH:EtOAc for characterisation,whilst the bulk was used without further purification]; ¹H NMR (400 MHz,DMSO): 12.99 (br s, 1H, pyrimidinyl-N⁺—H), 10.82 (s, 1H, ArNHAr), 10.76(br s, 1H, ArC(O)NHAr), 8.35 (dd, J=6.92, 1.99 Hz, 2H, ArH), 8.27 (d,J=6.92, 1.99 Hz, 2H, ArH), 7.90 (v v v br s, 2H, ArNH₂), 8.20-7.50 (m,4H, ArH & ArNH₂), 7.45-7.10 (m, 2H, ArH), 6.25 (s, 1H, ArH), 2.28 (s,3H, ArCH₃); LCMS (APCI⁺): 365 (100%).

4-Amino-N-[3-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]-benzamide(E4). To a refluxing suspension of amide E3 (7.89 g, 20.00 mmol) in 2:1EtOH:H₂O (500 mL) were sequentially added Fe dust (4.40 g, 79 mmol) andc.HCl (2% v/v, 10 mL), and the resulting mixture refluxed for ˜14 h.After this time, the hot reaction mixture was filtered through a pad ofCelite, and the solvent was removed under reduced pressure. The residuewas re-dissolved in hot H₂O, and the resulting suspension filteredthrough a pad of Celite. Solvent was removed under reduced pressure, andthe residue dried via three MeOH azeotrope cycles. The resulting residuewas reprecipitated from acidified MeOH:EtOAc to give amine E4 as anamorphous tan solid (0.95 g, 12%); ¹H NMR (400 MHz, DMSO): δ 12.71 (brs, 1H, pyrimidinyl-N⁺—H), 10.55 (br s, 1H, ArC(O)NHAr), 9.95 (s, 1H,ArNHAr), 8.10-7.40 (m, 7H, ArH & ArNH₂), 7.32 (m, 1H, ArH), 6.77 (d,J=8.30 Hz, 2H, ArH), 6.18 (s, 1H, ArH), 2.28 (s, 3H, ArCH₃); LCMS(APCI⁺): 335 (100%).

N-[3-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. II). To a solution of amine E4 (0.26 g, 0.63 mmol)in 1:2 EtOH:H₂O (30 mL) were sequentially added 4-chloroquinoline (0.62g, 3.78 mmol) and c.HCl (0.17 mL, 5.67 mmol), and the resulting mixturerefluxed for 3 h (reaction progress followed by TLC, eluting with thetop phase of a 5:4:1 mixture of n-BuOH:H₂O:acetic acid; productR_(f)=0.43, yellow spot after staining with KMnO₄). After this time,solvent was removed under reduced pressure, and the residue dried viathree MeOH-azeotrope cycles. The residue was re-precipitated fromMeOH:EtOAc to give Cpd. II as an amorphous lemon-yellow solid (0.27 g,79%); mp 251-255° C. (powder-glue); ¹H NMR (400 MHz, DMSO): δ 14.78 (vbr s, 1H, quinolinyl-N⁺H), 12.79 (v br s, 1H, pyrimidinyl-N⁺H), 11.14(s, 1H, ArNHAr), 10.64 (br s, 1H, ArC(O)NHAr), 10.52 (s, 1H, ArNHAr),8.87 (d, J=8.44 Hz, 1H, ArH), 8.61 (d, J=6.94 Hz, 1H, ArH), 8.30-7.45(m, 12H, ArH & ArNH₂), 7.38 (t, J=8.07 Hz, 1H, ArH), 7.00 (d, J=6.94 Hz,1H, ArH), 6.22 (s, 1H ArH), 2.29 (s, 31-1, ArCH₃); LCMS (APCI⁺): 463(100%); HPLC: 95.4%.

Example JJ Synthesis of(E)-N-[3-(1-{(diaminomethylene)hydrazono}ethyl)phenyl]-4-[6-(dimethylamino)quinolin-4-ylamino]benzamidedihydrochloride (Cpd. JJ 1) and the Related Compound (Cpd. JJ2)

(E)-N-[4-(1-{Diaminomethylene}hydrazono)ethyl]-3-nitrobenzenehydrochloride (F2). To a solution of aminoguanidine sulfate (21.25 g,80.41 mmol) and 3-nitroacetophenone (F1) (13.53 g, 81.93 mmol) in MeOH(400 mL) was added c.HCl (2.44 mL, 80.42 mmol), and the resultingmixture was refluxed for ˜14 h. Solvent was removed from the resultingwhite suspension to give diamine F2 as an amorphous white solid, whichwas used without further purification; ¹H NMR (400 MHz, DMSO): δ 11.00(v v br s, 1H, hydrazonyl-N⁺H), 8.59 (t, J=1.94 Hz, 1H, ArH), 8.39 (d,J=7.87 Hz, 1H, ArH), 8.19 (ddd, J=8.14, 2.18, 0.82 Hz, 1H, ArH), 7.83(br s, 4H, ═C(NH₂)₂)_(,) 7.66 (t, J=8.05 Hz, 1H, ArH), 2.40 (s, 3H,Ar(CH₃)═N—); LCMS (APCI⁺): 222 (100%).

(E)-N-[4-(1-{Diaminomethylene}hydrazono)ethyl]-3-benzaminedihydrochloride (F3). To a refluxing suspension of diamine F2 in 2:1EtOH:H₂O (500 mL) were sequentially added Fe dust (17.87 g, 320.00 mmol,4 molar equivalents) and c.HCl (10 mL), and the resulting mixturerefluxed for 14 h. After this time, the hot reaction mixture wasfiltered through a pad of Celite, and the solvent was removed underreduced pressure. The residue was re-dissolved in hot H₂O, and theresulting suspension filtered through a pad of Celite. Solvent wasremoved under reduced pressure, and the residue dried via threeMeOH-azeotrope cycles. The resulting residue was reprecipitated fromacidified MeOH/EtOAc to give triamine F3 (10.78 g, 51% over two steps)as an amorphous white solid; ¹H NMR (400 MHz, DMSO): δ 11.35 (s, 1H,hydrazonyl-N⁺H), 9.40 (v v br s, 3H, ArNH₃ ⁺), 7.84 (br m, 5H, ArH &═C(NH₂)₂), 7.75 (s, 1H, ArH), 7.43 (t, J=7.91 Hz, 1H, ArH), 7.27 (d,J=8.27 Hz, 1H, ArH), 2.34 (s, 3H, Ar(CH₃)═N—); LCMS (APCI⁺): 192 (100%).

(E)-N-[3-(1-{(Diaminomethylene)hydrazono}ethyl)phenyl]-4-nitrobenzamidedihydrochloride (F4). To a suspension of triamine F3 (5.15 g, 19.49mmol) and dry pyridine (7.85 mL, 97.45 mmol) in dry dioxane (300 mL) wasadded 4-nitrobenzoyl chloride (10.52 g, 56.69 mmol), and the resultingmixture refluxed for 17 h. After this time, the reaction mixture wascooled to room temperature, and then basified by addition of aqueousammonia solution. The resulting solution was diluted with H₂O, and theresulting precipitate collected by filtration to afford amide F4 (4.21g, 51%) as an amorphous creamy yellow solid; ¹H NMR (400 MHz, DMSO): δ11.09 (s, 1H, hydrazonyl-N⁺H), 10.68 (s, 1H, ArC(O)NHAr), 8.39 (d,J=9.22 Hz, 1H, ArH), 8.29 (m, 3H, ArH), 7.89 (d, J=8.07 Hz, 1H, ArH),7.82 (d, J=7.89 Hz, 1H, ArH), 7.73 (br s, 4H, ═C(NH₂)₂), 7.44 (t, J=7.98Hz, 1H, ArH), 2.34 (s, 3H, Ar(CH₃)═N—); LCMS (APCI⁺): 341 (100%).

(E)-4-Amino-N-[3-(1-{(diaminomethylene)hydrazono}ethyl)-phenyl]benzamidedihydrochloride (F5). To a refluxing suspension of amide F4 (2.02 g,5.36 mmol) in 2:1 EtOH:H₂O (100 mL) were sequentially added Fe dust(1.20 g, 21.44 mmol) and c.HCl (2 mL), and the resulting suspension wasrefluxed for 14 h. After this time, the hot reaction mixture wasfiltered through a pad of Celite, and the solvent was removed underreduced pressure. The residue was re-dissolved in hot H₂O, and theresulting suspension filtered through a pad of Celite. Solvent wasremoved under reduced pressure, and the residue dried via threeMeOH-azeotrope cycles. The resulting residue was reprecipitated fromacidified MeOH to afford triamine F5 (2.18 g, quantitative) as anamorphous cream solid; ¹H NMR (400 MHz, DMSO): δ 11.30 (s, 1H,hydrazonyl-N⁺H), 10.05 (s, 1H, ArC(O)NHAr), 8.21 (s, 1H, ArH), 8.20-7.55(m, 8H, ArH & ═C(NH₂)₂), 7.36 (t, J=7.90 Hz, 1H, ArH), 6.91 (d, J=7.96Hz, 1H, ArH), 5.30 (v br s, 3H, ArNH₃ ⁴), 2.35 (s, 3H, Ar(CH₃)═N—); LCMS(APCI⁺): 311 (100%).

(E)-N-[3-(1-{(Diaminomethylene)hydrazono}ethyl)phenyl]-4-[6-(dimethylamino)quinolin-4-ylamino]benzamidedihydrochloride (Cpd. JJ 1). To a solution of triamine F5 (0.22 g, 0.59mmol) in 1:2 EtOH:H₂O (60 mL) were sequentially added6-dimethylamino-4-chloroquinoline (0.13 g, 0.64 mmol) and c.HCl (0.16mL, 5.32 mmol), and the resulting solution was refluxed for 20 h(reaction progress followed by TLC, eluting with the top phase of a5:4:1 mixture of n-BuOH:H₂O:acetic acid). After this time, solvent wasremoved under reduced pressure, and the residue dried via twoMeOH-azeotrope cycles. The residue was re-precipitated from MeOH:EtOActo give Cpd. JJ1 (61 mg, 19%) as an amorphous dark brown solid; mp239-243° C. (powder→glue), 278-282° C. (glue→liquid); ¹H NMR (400 MHz,DMSO): δ 14.44 (s, 1H, quinolinyl-N⁺H), 11.20 (s, 1H, hydrazonyl-N⁺H),10.58 (s, 1H, ArC(O)NHAr), 10.46 (s, 1H, ArNHAr), 8.35 (d, J=6.71 Hz,1H, ArH), 8.26 (t, J=1.73 Hz, 1H, ArH), 8.22 (d, J=8.61 Hz, 3H, ArH),7.95 (m, 2H, ArH), 7.79 (m, 5H, ArH & ═C(NH₂)₂), 7.69 (d, J=2.52 Hz, 1H,ArH), 7.66 (d, J=8.61 Hz, 2H, ArH), 7.58 (d, J=2.43 Hz, 1H, ArH), 7.43(t, J=7.98 Hz, 1H, ArH), 6.95 (d, J=6.71 Hz, 1H, ArH), 3.15 (s, 6H,ArN(CH₃)₂), 2.37 (s, 3H, Ar(CH₃)═N—); LCMS (APCI⁺): 482 (100%); HPLC:95.8%.

(E)-N-[3-(1-{(Diaminomethylene)hydrazono}ethyl)phenyl]-4-(quinolin-4-ylamino)benzamidedihydrochloride (Cpd. JJ2). To a solution of triamine F5 (0.35 g, 0.91mmol) in 1:2 EtOH:H₂O (30 mL) were sequentially added 4-chloroquinoline(0.64 g, 3.90 mmol) and c.HCl (0.25 mL, 8.24 mmol), and the resultingsolution was refluxed for 20 h (reaction progress followed by TLC,eluting with the top phase of a 5:4:1 mixture of n-BuOH:H₂O:acetic acid;product R_(f)=0.51, yellow spot after staining with KMnO₄). After thistime, solvent was removed under reduced pressure, and the residue driedvia two MeOH-azeotrope cycles. The residue was re-precipitated fromMeOH:EtOAc to give Cpd. JJ2 (91 mg, 20%) as a pale yellow amorphoussolid; mp 240-244° C. (powder→glue), 264-268° C. (glue→liquid); ¹H NMR(400 MHz, DMSO): δ 14.67 (br s, 1H, quinolinyl-N⁺—H), 11.19 (s, 1H,hydrazonyl-N⁺H), 11.07 (s, 1H, ArC(O)NHAr), 10.48 (s, 1H, ArNHAr), 8.72(d, J=8.52 Hz, 1H, ArH), 8.61 (d, J=6.90 Hz, 1H, ArH), 8.24 (m, 3H,ArH), 8.08 (m, 2H, ArH), 7.95 (d, J=8.10 Hz, 1H, ArH), 7.82 (m, 6H, ArH& ═C(NH₂)₂), 7.68 (d, J=8.38 Hz, 2H, ArH), 7.43 (t, J=7.97 Hz, 1H, ArH),7.01 (d, 0.1=6.90 Hz, 1H, ArH), 2.37 (s, 3H, Ar(CH₃)₂═N—); LCMS (APCI⁺):438 (100%); HPLC: 96.4%.

Example KK Synthesis ofN-[4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(6,7,8-trifluoroquinolin-4-ylamino)benzamidedihydrochloride (Cpd. KK)

6,7,8-Trifluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (G2). Asolution of trifluoroquinolone ester G1 (3.82 g, 14.09 mmol) wasrefluxed in 1M NaOH for 14 h, and then cooled to room temperature. Thesolution was acidified with 1M NaOH, and the resulting suspensionfiltered to give trifluoroquinolone acid G2 (3.31 g, 97%) as anamorphous white solid, which was used without further purification; mp264-268° C.; ¹H NMR (400 MHz, DMSO): δ 14.20 (v br s, 2H, quinolinyl-N⁺H& ArCO₂H), 8.71 (s, 1H, ArH), 8.08 (ddd, J_(H—F)=10.17, 7.77, 2.23 Hz,1H, ArH); LCMS (APCI⁺): 244 (100%).

6,7,8-Trifluoroquinolin-4(1H)-one (G3). A solution of trifluoroquinoloneacid G2 (1.32 g, 5.43 mmol) was refluxed in biphenyl ether (100 mL) for30 min. After this time, the hot reaction mixture was poured carefullyinto hexanes, and resulting suspension was allowed to cool to roomtemperature. The suspension was filtered to give trifluoroquinolone G3(two batches, 1.32 g total, quantitative) as a very fine amorphousgrey-white solid, which was used without further purification; ¹H NMR(400 MHz, DMSO): δ 12.14 (s, 1H, ArOH), 7.91 (dd, J=6.73, 6.36 Hz, 1H,ArH), 7.80 (ddd, J_(H—F)=10.46, 8.11, 2.14 Hz, 1H, ArH), 6.10 (d, J=7.43Hz, 1H, ArH); LCMS (APCI⁺): 200 (100%).

4-Chloro-6,7,8-trifluoroquinoline (G4). A solution of quinolone G3 (1.30g, 6.53 mmol) was refluxed in POCl₃ (50 mL) for 1.3 h, and then excessPOCl₃ was removed under reduced pressure. The residue was re-dissolvedin CH₂Cl₂, and the resulting solution basified by addition of aqueousammonia solution. The resulting solution was extracted with CH₂Cl₂, andthe combined organic extracts washed sequentially with H₂O and brine,and dried over MgSO₄. Solvent was removed under reduced pressure, andthe residue purified by flash chromatography on silica gel, eluting with100% CH₂Cl₂→1% MeOH:CH₂Cl₂→10% MeOH:CH₂Cl₂), to give trifluoroquinolineG4 (0.76 g, 54%) as a white crystalline solid; mp 119-121° C.; ¹H NMR(400 MHz, DMSO): δ 8.93 (d, J=4.73 Hz, 1H, ArH), 8.08 (ddd,J_(H—F)=10.23, 7.92, 2.30 Hz, 1H, ArH), 7.95 (d, J=4.73 Hz, 1H, ArH);LCMS (APCI⁺): 218 (100%), 220 (100%).

N-[4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(6,7,8-trifluoroquinolin-4-ylamino)benzamidedihydrochloride (Cpd. KK). To a solution of amine G5 (0.19 g, 0.46 mmol)in dry MeOH (20 mL) were sequentially added trifluoroquinoline G4 (0.22g, 1.03 mmol) and c.HCl (˜3 drops), and the resulting solution wasrefluxed for 24 h (reaction progress followed by TLC, eluting with thetop phase of a 5:4:1 mixture of n-BuOH:H₂O:acetic acid; productR_(f)=0.56, yellow spot after staining with KMnO₄. This is same R_(f) asG5, however, so consumption of G5 was confirmed via LCMS analysis).After this time, solvent was removed under reduced pressure, and theresidue dried via three MeOH-azeotrope cycles. The residue wastriturated sequentially with MeOH, EtOAc and hexanes, and then driedunder high-vacuum to give Cpd. KK (two batches, 214 mg total, 79%) as anamorphous pale/lemon-yellow solid; mp>280° C.; ¹H NMR (400 MHz, DMSO): δ12.70 (br s, 1H, pyrimidinyl-N—H⁺), 10.77 (br s, 1H, ArC(O)NHAr), 10.66(s, 1H, ArNHAr), 10.39 (s, 1H, ArNHAr), 8.84 (m, 1H, ArH), 8.59 (d,J=6.35 Hz, 1H, ArH), 8.14 (d, J=8.62 Hz, 2H, ArH), 8.06-7.64 (m, 6H, ArH& ArNH₂), 7.61 (d, J=8.62 Hz, 2H, ArH), 7.11 (d, J=6.35 Hz, 1H, ArH),6.19 (s, 1H, ArH), 2.28 (s, 3H, ArCH₃) [quinolinyl-N⁺H not visible];LCMS (APCI⁺): 517 (100%), 518 (40%); HPLC: 98.9%.

Example LL Synthesis ofN-[4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(6-chloroquinolin-4-ylamino)benzamide(Cpd. LL)

4,6-Dichloroquinoline (H2). A solution of 6-chloro-4-quinolone (H1)(1.48 g, 8.22 mmol) in POCl₃ (50 mL) was refluxed for 3.5 h, and thenexcess POCl₃ was removed under reduced pressure. The residue wasre-dissolved in CH₂Cl₂, and the resulting solution basified by additionof aqueous ammonia solution. The resulting solution was extracted withCH₂Cl₂, and the combined organic extracts washed sequentially with H₂Oand brine, and dried over MgSO₄. Solvent was removed under reducedpressure to give dichloroquinoline H2 (1.54 g, 95%) as an amorphouscreamy-white solid; mp 101-103° C.; R_(f)=0.83 (5% MeOH:CH₂Cl₂); ¹H NMR(400 MHz, DMSO): δ 8.88 (d, J=4.73 Hz, 1H, ArH), 8.21 (d, J=2.33 Hz, 1H,ArH), 8.14 (d, J=8.99 Hz, 1H, ArH), 7.91 (dd, J=8.99, 2.33 Hz, 1H, ArH),7.84 (d, J=4.73 Hz, 1H, ArH); LCMS (APCI⁺): 198 (100%), 200 (80%).

N-[4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl]-4-(6-chloroquinolin-4-ylamino)benzamide(Cpd. LL). To a solution of amine G5 (0.18 g, 0.44 mmol) in dry MeOH (40mL) were sequentially added dichloroquinoline H2 (0.17 g, 0.88 mmol) andc.HCl (a few drops), and the resulting mixture was refluxed for 2 h. TLCanalysis after this time (eluting with the top phase of a 5:4:1 mixtureof n-BuOH:H₂O:acetic acid) appeared to show H2 had been consumed whilstsome E4 remained, thus a further portion of H2 (0.17 g, 0.88 mmol) wasadded at 3 h. The resulting mixture was refluxed for 14 h, and with nochange visible by TLC analysis, solvent was removed under reducedpressure and the residue dried via three MeOH-azeotrope cycles. Theresidue was triturated with MeOH, and then dried under high-vacuum togive Cpd. LL (2 batches, 0.21 g total, 83%) as an amorphous yellowsolid; mp 250-254° C.; ¹H NMR (400 MHz, DMSO): δ 14.80 (v br s, 1H,pyrimidinyl-N—H⁺), 12.62 (br s, 1H, quinolinyl-N⁺—H), 11.07 (s, 1H,ArC(O)NHAr), 10.62 (s, 1H, ArNHAr), 10.44 (s, 1H, ArNHAr), 9.01 (d,J=1.50 Hz, 1H, ArH), 8.63 (d, J=6.90 Hz, 1H, ArH), 8.13 (m, 4H, ArH),8.05-7.45 (m, 8H, ArH & ArNH₂), 7.04 (d, J=6.90 Hz, 1H, ArH), 6.18 (s,1H, ArH), 2.28 (s, 3H, ArCH₃); LCMS (APCI⁺): 497 (100%), 499 (60%);HPLC: 99.7%.

Example MM Synthesis ofN-[4-(pyridin-4-ylamino)phenyl]-4-(quinolin-4-ylamino)benzamidedihydrochloride (Cpd. MM)

N-[4-(Pyridin-4-ylamino)phenyl]-4-(quinolin-4-ylamino)-benzamidedihydrochloride (Cpd. MM). To a suspension of amine I1 (0.45 g, 1.18mmol) in 1:10 MeOH:EtOH was sequentially added 4-chloroquinoline (0.97g, 5.91 mmol), c.HCl (3 mL), and H₂O (10 mL), and the resulting mixturewas refluxed for ˜14 h (reaction progress followed by TLC, eluting withthe top phase of a 5:4:1 mixture of n-BuOH:H₂O:acetic acid; productR_(f)=0.30, yellow spot after staining with KMnO₄). After thus time,solvent was removed under reduced pressure, and the residue dried viathree MeOH-azeotrope cycles. The residue was re-precipitated fromMeOH:EtOAc, and purified further by preparative HPLC, to give Cpd. MM(0.14 g, 32% brsm) as an amorphous lemon-yellow solid; mp: 148-151° C.(powder→glue), 171-174° C. (glue→liquid); ¹H NMR (400 MHz, DMSO): δ13.97 (br s, 2H, pyridinyl-N⁺H & quinolinyl-N⁺H), 10.83 (br s, 1H,ArC(O)NHAr), 10.49 (s, 1H, ArNHAr), 10.44 (s, 1H, ArNHAr), 8.71 (d,J=8.47 Hz, 1H, ArH), 8.63 (d, J=6.81 Hz, 1H, ArH), 8.27 (d, J=7.14 Hz,2H, ArH), 8.17 (d, J=8.51 Hz, 2H, ArH), 8.05 (m, 2H, ArH), 7.94 (d,J=8.82 Hz, 2H, ArH), 7.85 (ddd, J=8.32, 5.64, 2.51 Hz, 1H, ArH), 7.67(d, J=8.51 Hz, 2H, ArH), 7.36 (d, J=8.80 Hz, 2H, ArH), 7.08 (d, J=7.27Hz, 2H, ArH), 7.03 (d, J=6.81 Hz, 1H, ArH); LCMS (APCI⁺): 431 (50%), 433(100%); HPLC: 100%.

Example NN Synthesis of(E)-N-(4-(1-((diaminomethylene)hydrazono)ethyl)phenyl)-4-(6-(dimethylamino)quinolin-4-ylamino)benzamidedihydrochloride (Cpd. NN)

(E)-N-(4-(1-((diaminomethylene)hydrazono)ethyl)phenyl)-4-(6-(dimethylamino)quinolin-4-ylamino)benzamidedihydrochloride (Cpd. NN). To a solution of amine 0.11 (0.22 g, 0.56mmol) in 1:2 EtOH:H₂O (20 mL) was sequentially added a solution of6-(dimethylamino)-4-chloroquinoline (0.13 g, 0.61 mmol) in 1:2 EtON:H₂O(10 mL) and c.HCl (0.17 mL, 5.5 mmol). The resulting mixture wasrefluxed for a few hours, and then allowed to warm to room temperatureovernight. After this time (16 h), TLC analysis (eluting with the topphase of a 5:4:1 mixture of n-BuOH:H₂O:CH₃CO₂H) showed some 6 remainedin the reaction mixture, so another equivalent of J1 (0.22 g, 0.56 mmol)was added, and the mixture refluxed for a further 16 h. After this time,TLC analysis showed almost complete consumption of the quinoline, sosolvent was removed under reduced pressure, and the residue was driedvia three MeOH-azeotrope cycles. The residue was reprecipitated fromMeOH (acidified with 1.25 methanolic HCl):EtOAc, and further purifiedvia preparative HPLC, to give Cpd. NN as an amorphous yellow-brown solid(18 mg, 6%); mp (MeOH:EtOAc)>280° C.; ¹H NMR [(CD₃)₂SO]: δ 2.33 [s, 3H,ArC(CH₃)═N—], 3.11 [s, 6H, ArN(CH₃)₂], 7.00 (s, 1H, ArH], 7.41 [s, 1H,ArH], 7.57-7.90 [m, 8H, ArH & ═C(NH₂)₂], 7.99 [d, J=8.45 Hz, 2H, ArH],8.12 [d, J=7.01 Hz, 2H, ArH], 8.35 [br s, 1H, ArH], 9.86 [br s, 1H,ArH], 10.40 [s, 1H, ArH], 10.86 [br s, 1H, ArNHAr] {3 remainingexchangeable H signals not visible}; LCMS (APCI⁺): 482 (100%); HPLC:98.7%.

Example OO Synthesis of6-(dimethylamino)-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)anilino]-quinoliniumdichloride (Cpd. OO1) and the Related Compound (Cpd. OO2)

6-(Dimethylamino)-4-[4-({3-[(1-methyl-4-pyridiniumyl)amino]-benzoyl}amino)anilino]-quinoliniumdichloride Sg EPG 133(Cpd. OO1). To a solution of amine K1 (151 mg, 0.43mmol) in ethanol (28 mL) and water (14 mL) was added6-(dimethylamino)-4-chloro quinoline (107 mg, 0.52 mmol) in ethanol (5mL) and two drops of c.HCl. The reaction mixture was refluxed for 3days, diluted with EtOAc (150 mL), brought to boil and allowed it tocool to 20° C. The resulting precipitate was filtered, washed with moreEtOAc and dried to give a yellow solid 194 mg, which was purified byprep. HPLC (TFA/CH₃CN) and then recrystallized from MeOH/EtOAC to giveCpd. OO1 (80 mg, 33%); mp (MeOH/EtOAc) 17-173° C. (dec); ¹H NMR[(CD₃)₃SO] δ 13.88 (bs, 1H, N^(+H),) 10.64 (s, 1H, NH), 10.53 (s, 1H,NH), 10.25 (bs, 1H, NH), 8.32 (d, J=d, 0.1=7.5 Hz, 2H, ArH), 8.27 (d,J=6.5 Hz, 1H, ArH), 7.98 (d, J=8.9 Hz, 2H, ArH), 7.94 (d, J=8.0 Hz, 1H,ArH), 7.90 (bs, 1H, ArH), 7.84 (d, J=9.4 Hz, 1H, ArH), 7.69 (t, J=7.9Hz, 1H, ArH), 7.64 (dd, J=9.4, 2.6 Hz, 1H, ArH), 7.58 (dd, J=8.0, 1.3Hz, 1H, ArH), 7.48-7.45 (m, 3H, ArH), 7.22 (d, J=7.5 Hz, 2H, ArH), 6.68(s, 1H, ArH), 3.99 (s, 3H, N⁺CH₃), 3.12 [s, 6H, N(CH₃)₂]; APCI ve⁺ 489.

6-(Dimethylamino)-4-[4-({4-[(1-methyl-4-pyridiniumyl)amino]benzoyl}amino)-anilino]quinoliniumdichloride SG EPG 134 (Cpd. OO2). To a solution of amine K2 (162 mg,0.46 mmol) in ethanol (28 mL) and water (14 mL) was added6-(dimethylamino)-4-chloro quinoline (114 mg, 0.55 mmol) in ethanol (5mL) and two drops of c.HCl. The reaction mixture was refluxed for 2days, diluted with EtOAc (150 mL), brought to boil and allowed it tocool to 20° C. The resulting precipitate was filtered, washed with moreEtOAc and dried to give a yellow solid 161 mg, which was purified byprep. HPLC (TFA/CH₃CN) and then recrystallized from MeOH/EtOAC to giveCpd. OO2 (80 mg, 31%); mp (MeOH/EtOAc) 156° C. (dec); ¹H NMR [(CD₃)₂SO]δ 13.90 (bs, 1H, N⁺H), 10.73 (s, 1H, NH), 10.48 (s, 1H, NH), 10.29 (bs,1H, NH), 8.36 (d, J=7.5 Hz, 2H, ArH), 8.28 (d, J=6.7 Hz, 1 H, ArH), 8.11(d, J=8.6 Hz, 2H, ArH), 8.00 (d, J=8.8 Hz, 2H, ArH), 7.84 (d, J=9.4 Hz,1H, ArH), 7.65 (dd, J=9.4, 2.2 Hz, 1H, ArH), 7.52-7.45 (m, 5H, ArH),7.28 (d, J=7.5 Hz, 2H, ArH), 6.68 (d, J=6.8 Hz, 1H, ArH), 4.01 (s, 3H,N⁺CH₃), 3.12 [s, 6H, N(CH₃)₂] APCI ve⁺ 489.

Example PP Preparation of4-[(1E)-N-(diaminomethylene)ethanehydrazonoyl]-N-(4-{[6-(dimethylamino)-4-quinolinyl]amino}phenyl)benzamidedihydrochloride (Cpd. PP)

4-[(1E)-N-(Diaminomethylene)ethanehydrazonoyl]benzoic acid hydrochloride(L2). 4-Acetylbenzoic acid (L1) (1.041 g, 6.34 mmol), amino guanidinebicarbonate (1.12 g, 8.2 mmol, 1.3 eq) and c.HCl (0.7 ml, 7.0 mmol) inMeOH (30 mL) was refluxed for 1 h. The reaction mixture was diluted withEtOAc cooled to 20° C. and the resulting precipitate was filtered andrecrytallized from MeOH/EtOAc to give L2 (887 mg, 55%), mp(MeOH/EtOAc)>300° C.; ¹H NMR ([(CD₃)₂SO] δ 7.97-7.89 (m, 4H, ArH), 6.75(br, 4H, 2×NH₂), 2.25 (s, 3H, CH₃), mass APCI⁺221.

4-[(1E)-N-(Diaminomethylene)ethanehydrazonoyl]-N-(4-{[6-(dimethylamino)-4-quinolinyl]amino}phenyl)benzamidedihydrochloride (Cpd. PP).N⁴-(4-Aminophenyl)-N⁶,N⁶-dimethyl-4,6-quinolinediamine (A6) (108 mg,0.34 mmol), L2 (107 mg, 0.34 mmol), EDCI (160 mg, 0.68 mmol) and DMAP(101 mg, 0.68 mmol) in DMF (10 mL) were stirred at 20° C. for 72 h. Thesolvent was evaporated under reduced pressure at 55° C. The residue wasdiluted with water and basified with aq NH₃. The resulting precipitatewas filtered washed with water, air dried and chromatographed(SiO₂/DCM/MeOH/aq NH₃ 0-7% 2% NH₃). The fractions containing correctmass were combined and evaporated to dryness to give 120 mg of a yellowsolid. This was converted to HCl salt by adding few drops of 4N HCl in1,4-dioxane to a suspension in MeOH, then the solvent was evaporated todryness. The resulting residue was recrystallized from MeOH/EtOAc togive a crude product (107 mg) containing two main compounds by HPLC.This was purified by prep. HPLC(HCOO⁻N⁺H₄) to give Cpd. PP (52 mg, 27%);m.p (MeOH/EtOAc)>300° C.; ¹H NMR [(CD₃)₂SO] δ 14.09 (br s, 1H, N⁺H),11.21 (s, 1H, NH), 10.56 (s, 1H, NH), 10.40 (s, 1H, NH), 8.27 (d, J=6.8Hz, 1H, ArH), 8.13 (d, J=8.6 Hz, 2H, ArH), 8.06-8.01 (m, 4H, ArH), 7.88(d, J=9.4 Hz, 1H, ArH), 7.83 (br, 4H, 2×NH₂), 7.65 (dd, J=9.4, 2.3 Hz,1H, ArH), 7.53 (d, J=2.3 Hz, 1H, ArH), 7.47 (d, J=8.9 Hz, 2H, ArH), 6.67(d, J=6.8 Hz, 1H, ArH), 3.13 [s, 6H, [N(CH₃)₂], 2.41 (s, 3H, CH₃); massAPCI⁺ 481.

Example QQ Preparation of4-[4-({3-[(1E)-N-(diaminomethylene)ethanehydrazonoyl]-benzoyl}amino)anilino]-6-(dimethylamino)quinoliniumchloride (Cpd. QQ)

4-{4-[(3-acetylbenzoyl)amino]anilino}-6-(dimethylamino)-quinoliniumchloride (M2). A mixture ofN^(∝)-(4-Aminophenyl)-N⁶,N⁶-dimethyl-4,6-quinolinediamine (181 mg, 0.58mmol), 3-acetylbenzoic acid (M1) (97 mg, 0.58 mmol) and EDCI (220 mg,0.1.16 mmol) in DMF (5 mL) was stirred at 20° C. for 5 min. Then DMAP(140 mg, 1.16 mmol) was added and the reaction mixture was stirred at20° C. for 24 h. The solvent was removed under reduced pressure andresidue was stirred in aqueous NaHCO₃ for 1 h. The resulting precipitatewas filtered and purified by chromatography in SiO₂ eluting with agradient (0-7.5%) of MeOH/DCM to give M2 (113 mg, 42%); mp(DCM/MeOH)>280° C.; ¹H NMR [(CD₃)₂SO] δ 14.02 (br, 1H, NH), 10.66 (s,1H, NH), 10.31 (s, 1H, NH), 8.53 (t, J=1.6 Hz, 1H, ArH), 8.28 (d, J=6.7Hz, 1H, ArH), 8.24 (td, J=8.1, 1.5 Hz, 1H, ArH), 8.19 (td, J=7.8, 2.8Hz, 1H, ArH), 8.00 (d, J=6.8 Hz, 2H, ArH), 7.87 (d, J=9.4 Hz, 1H, ArH),7.78 (t, J=7.8 Hz, 1H, ArH), 7.64 (dd, J=9.4, 2.6 Hz, 1H, ArH), 7.51 (d,J=2.5 Hz, 1H, ArH), 7.47 (d, J=8.8 Hz, 2H, ArH), 6.69 (d, J=6.7 Hz, 1H,ArH), 3.09 [s, 6H, (NCH₃)₂], 2.68 (s, 3H, COCH₃); APCI⁺ve 425.

4-[4-({3-[(1E)-N-(diaminomethylene)ethanehydrazonoyl]benzoyl}amino)anilino]-6-(dimethyl-amino)quinoliniumchloride (Cpd. QQ). A mixture of M2 (94 mg, 0.20 mmol), aminoguanidinebicarbonate (42 mg, 0.3 mmol) and c.HCl (0.02 mL, 0.022 mmol) in MeOH 10mL) was refluxed for 2 hr diluted with EtOAc, some of the MeOH wasboiled off and cooled to 20° C. The resulting precipitate was filtered,washed with more EtOAc and recrystallized from MeOH/EtOAc to give Cpd.QQ (109 mg 100%) as a yellow solid; mp (MeOH/EtOAC)>280° C.; ¹H NMR[(CD₃)₂SO] δ 14.13 (br 1H, N⁺H), 11.25 (s, 1H, NH), 10.67 (s, 1H, NH),10.42 (s, 1H, NH), 8.48 (t, J=1.5 Hz, 1 H, ArH), 8.27 (d, J=6.8 Hz, 1H,ArH), 8.22 (t, d, J=7.9, 1.4 Hz, 1H, ArH), 8.05-8.01 (m, 3H, ArH), 7.89(d, J=9.6 Hz, 1H, ArH), 7.85 (br, 4H, 2×NH₂), 7.66-7.59 (m, 2H, ArH),7.54 (d, J=2.3 Hz, 1 H, ArH), 7.47 (d, J=8.8 Hz, 2H, ArH), 6.67 (d,J=6.8 Hz, 1H, ArH), 3.13 [s, 3, 6H, (NCH₃)₂], 2.45 (s, 3H, CH₃); APCI⁺ve481.

Example RR Preparation of3-[(2,6-diamino-4-pyrimidinyl)amino]-N-{4-[(6-nitro-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. RR1) and the Related Compounds (Cpd. RR2 AND Cpd.RR3)

3-[(2,6-diamino-4-pyrimidinyl)amino]-N-{-4-[(6-nitro-4-quinolinyl)-amino]phenyl}benzamidedihydrochloride (Cpd. RR1). To a solution of amine B6 (204 mg, 0.55mmol) in EtOH (20 mL) and H₂O (10 mL) was added4-chloro-6-nitroquinoline (126 mg, 0.61 mmol) and stirred until itdissolved, then 2 drops of c.HCl was added. The reaction mixture wasrefluxed for 4 h, diluted with EtOAc, brought to boil and cool to 20° C.The resulting precipitate was filtered and recrystallized fromMeOH/EtOAc to give Cpd. RR1 (224 mg 70%); m.p. (MeOH/EtOAc)>300° C.; ¹HNMR [(CD₃)₂SO] δ 14.75 (br, 1H, N⁺H), 11.50 (br, 1H, N⁺H), 11.10 (br,1H, NH), 10.50 (s, 1H, NH), 9.88 (s, 1H, NH), 9.77 (s, 1H, NH), 9.76 (s,1H, ArH), 8.66 (d, J=9.3 Hz, 1H, ArH), 8.60 (d, J=6.8 Hz, 1H, ArH), 8.19(d, J=9.3 Hz, 1H, ArH), 8.25 (d, J=8.8 Hz, 2H, ArH), 7.94 (br s 2H,NH₂), 7.71 (d, J=7.6 HZ, 1H, ArH), 7.56-7.42 (m, 6H, NH₂ & 4×ArH), 6.91(d, J=6.8 Hz, 1H, ArH), 5.44 (s, 1H, ArH). HRMS (FAB⁺) calc. forC₂₆H₂₂N₉O₃ (M⁺¹) m/z 508.1846, found 508.1841.

3-[(2-amino-6-methyl-4-pyrimidinyl)amino]-N-{4-[(6-nitro-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. RR2). To a solution of amine B3 (205 mg, 0.55mmol) in EtOH (20 mL) and H₂O (10 mL) was added4-chloro-6-nitroquinoline (135 mg, 0.64 mmol) and stirred until itdissolved, then 2 drops of c.HCl was added. The reaction mixture wasrefluxed for 5 h, diluted with EtOAc, brought to boil and cool to 20° C.The resulting precipitate was filtered and recrystallized fromMeOH/EtOAc to give Cpd. RR2 (217 mg 68%); m.p. (MeOH/EtOAc)>300° C.; ¹HNMR [(CD₃)₂SO] δ 13.00 (br, 2H, 2×N⁺H), 10.99 (br, 1H, NH), 10.75 (br s,1H, NH), 10.53 (s, 1H, NH), 9.75 (d, J=1.9 Hz, 1H, ArH), 8.64 (br d,J=7.4 Hz, 1H, ArH), 8.60 (d, J=6.7 Hz, 1H, ArH), 8.17 (d, J=9.3 Hz, 1H,ArH), 8.15 (br, 1H, NH), 8.07 (br s, 1H, NH), 7.99 (d, J=8.8 Hz, 2H,ArH), 7.79 (br d, J=7.5 Hz, 3H, ArH), 7.57 (t, J=7.9 Hz, 1H, ArH), 7.48(d, J=8.8 Hz, 2H, ArH), 6.91 (d, J=6.7 Hz, 1H, ArH), 6.22 (d, J=0.6 Hz,1H, ArH), 2.30 (s, 3H. CH₃); HRMS (FAB⁺) calc. for C₂₇H₂₃N₈O₃ (M⁺¹) m/z507.1893, found 507.1888.

3-[(2-Amino-6-methyl-4-pyrimidinyl)amino]-N-{4-[(6-amino-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. RR3). Compound Cpd. RR2 (146 mg, 146 mg, 0.25mmol) was dissolved in MeOH (30 ml) and hydrogenated with 10% Pd/C (20mg) at 30 Hg mm for 3 h. The reaction mixture was filtered through a padof celite. The filtrate was evaporated to dryness. The resulting residuewas dissolved in MeOH (10 mL) stirred with 1.25 M HCl in MeOH (0.5 mL),then precipitated by adding EtOAc, filtered and dried to give 120 mg ofCpd. RR3. This was 90% clean by HPLC. This was then converted to freebase by stirring in aq NH₃ filtered, air dried and chromatographed onneutral alumina eluting with a gradient (0-10%) of MeOH/DCM containing1% aq NH₃ to give 83 mg of clean free base. This was then converted toHCl salt by dissolving in MeOH and adding 1.25 M HCl in MeOH. Evaporatedto dryness and the residue was recrystallized from MeOH/EtOAc to giveCpd. RR3 (88 mg, 64%), mp (MeOH/EtOAc) 280-284° C.; ¹H NMR [(CD₃)₂SO] δ14.0 (d, J=5.4 Hz, 1H, N⁺H), 12.78 (br s, 1H, N⁺H), 10.79 (br s, 1H,NH), 10.50 (s, 1H, NH), 10.19 (s, 1 H, NH), 8.22 (t, J=6.4 Hz, 2H, ArH),8.16-8.07 (br, 2H, NH₂) 7.96 (d, J=8.9 Hz, 2H, ArH), 7.80 (br s, 1H,ArH), 7.78 (d, J=9.0 Hz, 2H, ArH), 7.56 (t, J=8.0 Hz, 1H, ArH), 7.47 (brd, J=2.1 Hz, 1 ArH), 7.43 (d, J=8.7 Hz, 2H, ArH), 7.39 (dd, J=9.0, 2.2Hz, 1H, ArH), 6.67 (d, J=6.8 Hz, 1H, ArH), 6.23 (s, 1H, ArH), 5.9 (v.br, 2H, NH₂), 2.31 (s, 3H, CH₃).

Example SS Preparation of4-[(2,6-diamino-4-pyrimidinyl)amino]-N-(4-{[6-(dimethylamino)-4-quinolinyl]amino}phenyl)benzamidedihydrochloride (Cpd. SS)

4-[(2,6-Diamino-4-pyrimidinyl)amino]benzoic acid (O1). 4-amino-benzoicacid (D1) (2.0 g, 14.55 mmol), and 2,6-diamino-4-chloropyrimidine (B4)(2.013 g, 14.55 mmol) were dissolved in 2-ethoxyethanol (20 mL). 2 dropsof c. HCl was added to this mixture and refluxed for 20 h. The reactionmixture was cooled to 20° C. and the resulting precipitate was filteredand recrystallized from MeOH/EtOAc to give compound O1 (3.12 g, 56%), mp(MeOH/EtOAc)° C.; ¹H NMR [(CD₃)₂SO] δ 12.65 (br, 1H, COOH or N⁺H), 11.84(br s, 1H, N⁺H or COOH), 10.07 (s, 1H, NH), 7.86 (br d, J=8.7 Hz, 2H,ArH), 7.75 (v.br d, J=7.8, 2H, ArH), 7.64 (br, 2H, NH₂), 7.51 (br, 2H,NH₂), 5.50 (s, 1H, ArH).

4-[(2,6-Diamino-4-pyrimidinyl)amino]-N-(4-{[6-(dimethylamino)-4-quinolinyl]amino}phenyl)benzamidedihydrochloride (Cpd. SS). Compound 01 (101.4 mg, 0.36 mmol), EDCI (138mg, 0.72 mmol) and DMAP (88 mg, 0.36 mmol) in N-methylpyrrolidinone (5mL) was stirred at 20° C. for 5 min. ThenN⁴-(4-Aminophenyl)-N⁶,N⁶-dimethyl-4,6-quinolinediamine (100 mg, 0.36mmol) and Et₃N (0.2 mL, 1.44 mmol) was added and stirred 20 h. The ticof a small sample (Al₂O₃/DCM/MeOH 5% and/aqNH₃) showed still presence ofN⁴-(4-Aminophenyl)-N⁶,N⁶-dimethyl-4,6-quinolinediamine, therefore moreEDCI (138 mg 0.72 mmol) was added and stirred 72 h. The reaction mixturewas then diluted with H₂O and stirred for 1 h. The resulting precipitatewas filtered, washed with water, air dried and chromatographed onneutral alumina eluting with a gradient of 0-5% of DCM/MeOH, to removeimpurity of unreactedN⁴-(4-Aminophenyl)-N⁶,N⁶-dimethyl-4,6-quinolinediamine then adding 1%aq.NH₃ to elute the product Cpd. SS. Evaporation of the fractionscontaining the product gave compound Cpd. SS (75 mg). This was dissolvedin a small amount of MeOH and stirred with 1.25 M HCl in MeOH (0.5 mL),solvent was evaporated and the residue recrystallized from MeOH/EtOAc togive Cpd. SS (84 mg, 40%), mp (MeOH/EtOAc) 283-287° C.; ¹H NMR[(CD₃)₂SO] δ 14.14 (d, J=4.7 Hz, 1H, N⁺H), 11.81 (br, 1H, N⁺H), 10.42(s, 1H, NH), 10.39 (s, 1H, NH), 10.09 (s, 1H, NH), 8.26 (t, J=6.4 Hz 1H,ArH), 8.02-7.98 (m, 4H, ArH), 7.89 (d, J=8.7 Hz, 1H, ArH), 7.80 (br d,2H, ArH), 7.68 (br, 2H, NH₂), 7.64 (dd, J=9.4, 2.5 Hz, 1H, ArH), 7.53(dd, J=9.3, 2.5 Hz, 1H, ArH), 7.52 (br s, 2H, NH₂), 7.45 (d, J=8.9 Hz,2H, ArH), 6.66 (d, J=6.8 Hz, 1 H, ArH), 5.51 (s, 1H, ArH), 3.12 [s, 6H,N(CH₃)₃].

Example TT Preparation of4-[(2,6-diamino-4-pyrimidinyl)amino]N-[4-(4-quinolinylamino)phenyl]benzamidedihydrochloride (Cpd. TT)

4-[(2,6-Diamino-4-pyrimidinyl)amino]-N-(4-nitrophenyl)-benzamidehydrochloride (P2). Amine PI (1.0 g, 3.89 mmol) and chloropyrimidine B4(1.12 g, 7.78 mmol) were dissolved in MeOH (200 mL) by heating then c.HCl (3 drops) were added and refluxed for 5 days. The reaction mixturewas cooled to 20° C. and the precipitate was filtered washed with moreMeOH and dried to give essentially pure compound P2 (814 mg 52%), ¹H NMR[(CD₃)₂SO] δ 11.84 (s, 1H, N⁺H), 10.75 (s, 1H, NH), 10.12 (s, 1H, NH),8.28-8.24 (m, 2H, ArH), 8.11-8.07 (m, 2H, ArH), 8.24 (br d, J=8.7 Hz,2H, ArH), 7.83 (br, 2H, ArH), 7.68 (br, 2H, NH₂), 7.54 (br, 2H, NH₂),5.51 (s, 1H, ArH). HRMS (FAB⁺) calc. for C₁₅H₁₆N₇O₃ (M⁺¹) m/z 366.1315,found 366.1306.

N-(4-Aminophenyl)-4-[(2,6-diamino-4-pyrimidinyl)amino]-benzamidedihydrochloride (P3). A suspension of compound P2 (811 mg, 2.01 mmol) inMeOH 100 (mL) was hydrogenated with 10% Pd/C (100 mg) at 40 Hg mm H₂pressure for 20 h. The resulting new suspension was stirred with 1.25 MHCl in MeOH (5 mL) to dissolve the product and this was then filteredthrough a pad of Celite to remove Pd residues. The filtrate wasevaporated to dryness and the residue was recrystallized from MeOH/EtOActo give compound P3 (785 mg, 100%), ¹H NMR [(CD₃)₂SO] δ 10.02 (s, 1H,NH), 9.88 (s, 1H, NH), 7.91 (d, J=8.7 Hz, 2H, ArH), 7.74 (d, J=8.5 Hz,2H, ArH), 7.62 (br, 2H, NH₂), 7.68 (br s, 2H, NH₂), 7.47 (d, J=8.8 Hz,2H, ArH), 6.71 (d, J=8.7 Hz, 2H, ArH), 5.45 (s, 1H, ArH).

4-[(2,6-Diamino-4-pyrimidinyl)amino]-N-[4-(4-quinolinylamino)-phenyl]benzamidedihydrochloride (Cpd. TT). To a solution of compound P3 (128 mg, 0.34mmol) in EtOH (20 mL) and H₂O (10 mL) was added 4-chloroquinoline (127mg, 0.51 mmol) and stirred until it dissolved, then 2 drops of c.HCl wasadded. The reaction mixture was refluxed for 4 h, diluted with EtOAc,brought to boil and cool to 20° C. The resulting precipitate wasfiltered and recrystallized from MeOH/EtOAc to give Cpd. TT (151 mg,83%); m.p. (MeOH/EtOAc) 263-267° C.; ¹H NMR [(CD₃)₂SO] δ 14.10 (br, 1H,N⁺H), 11.90 (br, 1H, N⁺H), 10.89 (s, 1H, NH), 10.40 (s, 1H, NH), 10.04(s, 1H, NH), 8.77 (d, J=8.7 Hz, 1H, ArH), 8.51 (d, J=7.0 Hz, 1H, ArH),8.06-7.98 (m, 6H, ArH), 7.83-7.79 (m, 3H, ArH & NH₂), 7.61 (br, 2H,NH₂), 7.47 (d, J=8.9 Hz, m, 2H, ArH), 6.78 (d, J=7.0 Hz, 1H, ArH), 5.45(s, 1 H, ArH).

Example UU Preparation ofN-{4-[(6-amino-4-quinolinyl)amino]phenyl}-3-[(2,6-diamino-4-pyrimidinyl)amino]benzamidedihydrochloride (Cpd. UU)

N-{4-[(6-Amino-4-quinolinyl)amino]phenyl}-3-[(2,6-diamino-4-pyrimidinyl)amino]benzamidedihydrochloride (Cpd. UU).3-[(2,6-Diamino-4-pyrimidinyl)amino]-N-{4-[(6-nitro-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. RR1) (148 mg, 0.25 mmol) was dissolved in MeOH (30ml) and hydrogenated with 10% Pd/C (20 mg) at 30 Hg mm for 20 h. Thereaction mixture was filtered through a pad of celite. The filtrate wasevaporated to dryness. The resulting residue was dissolved in MeOH (10mL) stirred with 1.25 M HCl in MeOH (0.5 mL), then precipitated byadding EtOAc, filtered and dried to give 90 mg of compound UU. This was78% clean by HPLC. This was then converted to free base by stirring inaq NH₃ filtered, air dried and chromatographed on neutral aluminaeluting with a gradient (0-7%) of MeOH/DCM containing 1.5% aq NH₃ togive 51 mg of clean free base of Cpd. UU (51 mg, 43%). This was thenconverted to HCl salt by dissolving in MeOH and adding 1.25 M HCl inMeOH. Evaporated to dryness and the residue was recrystallized fromMeOH/EtOAc to give Cpd. UU, mp (MeOH/EtOAc) 250-255° C.; ¹H NMR[(CD₃)₂SO] δ 14.15 (d, J=6.1 Hz, 1H, N⁺H), 11.70 (br 1H, N⁺H), 10.48 (s,1H, NH), 10.46 (s, 1H, NH, 10.21 (S, 1H, NH), 8.22 (t, J=6.5 Hz, 1H,ArH), 8.05-7.89 (m, 4H, ArH), 7.79 (d, J=9.1 Hz, 1H, ArH), 7.71 (br d,J=7.5 Hz, 1H, ArH), 7.59 (br, 2H, NH₂), 7.53-7.38 (m, 8H, ArH & NH₂),6.67 (d, J=6.7 Hz, 1H, ArH), 5.45 (s, 1H, ArH). HPLC purity %. HRMS(FAIT), calc. for C₂₆H₂₃N₉O (M⁺¹) m/z 478.2104, found, 478.2103.

Example VV Preparation ofD[(2,6-diamino-4-pyrimidinyl)amino]-N-{4-[(6-nitro-4-quinolinyl)amino]phenyl}benzamidedihydrochloride (Cpd. VV1) and the Related Compound (Cpd. VV2)

4-[(2,6-Diamino-4-pyrimidinyl)amino]-N-{4-[(4-nitro-4-quinolinyl)-amino]phenyl}benzamidedihydrochloride (Cpd. VV1). To a solution of compound P3 (288 mg, 0.76mmol) in EtOH (20 mL) and H₂O (10 mL) was added4-chloro-6-nitroquinoline (196 mg, 0.94 mmol) and stirred until itdissolved, then 2 drops of c.HCl was added. The reaction mixture wasrefluxed for 4 h, diluted with EtOAc, brought to boil and cool to 20° C.The resulting precipitate was filtered and recrystallized fromMeOH/EtOAc to give Cpd. VV1 (445 mg 99%); m.p. (MeOH/EtOAc) 260-265° C.;¹H NMR [(CD₃)₂SO] δ 11.38 (br s, 1H, N⁺H), 10.43 (s, 1H, NH), 10.10 (s,H1, NH), 9.81 (d, J=2.1 Hz, 1H, NH), 8.78 (dd, J=9.3, 2.2 Hz, 1H, ArH),8.84 (d, J=7.2 Hz, 1H, ArH), 8.24 (d, J=9.3 Hz, 1H, NH), 8.04-7.98 (m,4H, ArH), 7.81 (br d, J=7.4 Hz, 2H, ArH), 7.68 (br s, 2H, NH₂), 7.53 (brs, 2H, NH₂), 7.48 (d, J=8.9 Hz, 2H, ArH), 6.90 (d, J=7.0 Hz, 1H, ArH),5.52 (s, 1H, ArH). HPLC purity 100%; HRMS (FAB⁺) calc for C₂₆H₂₁N₉O₃(M⁺¹) m/z 508.1846, found 508.1844; Anal. Calc. for C₂₆H₂₃Cl₂N₉O₃.4H₂O:C, 47.9; H, 4.2; N, 19.3; Cl, 10.9; found C, 48.0; H, 4.4; N, 19.2; Cl,11.1%.

N-{4-[(6-Amino-4-quinolinyl)amino]phenyl}-4-[(2,6-diamino-4-pyrimidinyl)amino]benzamidedihydrochloride (Cpd. VV2). A solution of Cpd. VV1 (211 mg, 0.36 mmol)was dissolved in MeOH (30 ml) and hydrogenated with 10% Pd/C (20 mg) at30 Hg mm for 5 h. The reaction mixture was filtered through a pad ofcelite. The filtrate was evaporated to dryness. The resulting residuewas dissolved in MeOH (10 mL) stirred with 1.25 M HCl in MeOH (0.5 mL),then MeOH was evaporated to dryness. Residue was redissolved in MeOH andevaporated to dryness, and then recrystallized from MeOH/EtOAc to give178 mg of the product; this was only 93% clean by HPLC. This was thenconverted to free base by stirring in aq NH₃ filtered, air dried andchromatographed on neutral alumina eluting with a gradient (0-7.5%) ofMeOH/DCM containing 1.5% aq NH₃ to give clean free base of Cpd. VV2.This was then converted to HCl salt by dissolving in MeOH and adding1.25 M HCl in MeOH. Evaporated to dryness and the residue wasrecrystallized from MeOH/EtOAc to give Cpd. VV2 (138 mg, 70%), mp(MeOH/EtOAc) 262-266° C.; ¹H NMR [(CD₃)₂SO] δ 14.11 (br s, 1H, N⁺H),11.81 (br, 1H, N⁺H), 10.36 (s, 1H, NH), 10.19 (s, 1H, NH), 10.08 (s, 1H,NH), 8.21 (t, J=6.0 Hz, 1H, ArH), 8.00-7.96 (m, 4H, ArH), 7.79 (br s,2H, NH₂), 7.78 (d, J=9.1 Hz, 1H, ArH), 7.67 (br s, 2H, NH₂), 7.53 (br s,2H, NH₂), 7.48 (br s, 1H, ArH), 7.42-7.38 (m, 3H, ArH), 6.66 (d, J=6.7Hz, 1H, ArH), 6.00 (v.br, 2H, NH₂), 5.51 (s, 1H, ArH). HPLC purity99.7%; HRMS (FAB⁺) calc. for C₂₆H₂₄N₉O (M⁺¹) m/z 478.2104 found478.2107.

Example WW Preparation ofN-methyl-N-[4-(pyridin-4-ylamino)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. WW)

N-Methyl-4-nitro-N-[4-(pyridin-4-ylamino)phenyl]benzamide hydrochloride(3). To a solution of amine 1 (0.82 g, 4.10 mmol) in dry dioxane (70 mL)were sequentially added dry pyridine (1.65 mL, 20.50 mmol) and acidchloride 2 (2.08 g, 11.19 mmol), and the resulting mixture was refluxedfor ˜60 h. After this time, the reaction mixture was cooled to roomtemperature, and the resulting solid collected by filtration. Thefiltrate was basified by addition of aqueous NH₃, and the resultingsecond batch of solid collected by filtration. Batches of solid werecombined to give amide 3 as an amorphous yellow solid (3.46 g), whichwas analyzed by ¹H NMR and MS, and used without further purification. ¹HNMR: 8.88 (dd, J=6.32, 1.32 Hz, 4H, ArC(O)N(CH₃)Ar & ArNHAr), 8.32 (ddd,J=9.17, 4.31, 2.27 Hz, 4H, ArH), 8.17 (ddd, J=9.17, 4.31, 2.28), 7.98(dd, J=7.59, 6.50 Hz, 4H, ArH) [pyridyl-N⁺—H not visible]; LCMS (APCI⁺):349 (100%).

4-Amino-N-methyl-N-[4-(pyridin-4-ylamino)phenyl]benzamide hydrochloride(4). To a solution of amide 3 (3.46 g, 8.99 mmol) in MeOH (˜40 mL) wasadded a spatula tipful of 10% Pd/C, and the resulting suspensionhydrogenated at 40 psi for 16 h. After this time, the reaction mixturewas filtered through a pad of Celite, and solvent removed under reducedpressure. The residue was reprecipated from MeOH-EtOAc to give amine 4as an amorphous cream solid (0.25 g, 15% from 1); mp 235-238° C.(powder-tar), 245-249° C. (gas evolved); ¹H NMR: 13.85 (v br s, 1H,pyridine-N⁺—H), 10.75 (s, 1H, ArNHAr), 8.28 (d, J=7.23 Hz, 2H, ArH),7.25 (s, 4H, ArH), 7.16 (d, J=8.44 Hz, 2H, ArH), 7.07 (d, J=7.03 Hz, 2H,ArH), 6.73 (d, J=7.79 Hz, 2H, ArH), 3.36 (s, ArC(O)N(CH₃)Ar) [ArN⁴H₃ notvisible]; HRMS (EI) calc. for C₁₉H₁₈N₄O m/z 318.1481, found 318.1481.

N-Methyl-N-[4-(pyridin-4-ylamino)phenyl]-4-(quinolin-4-ylamino)-benzamidehydrochloride Cpd. WW. To a solution of amine 4 (0.23 g, 0.58 mmol) in20% aqueous EtOH (40 mL) were sequentially added quinoline 5 (0.22 g,1.36 mmol) and c.HCl (0.17 mL, 5.60 mmol), and the resulting mixture wasrefluxed for ˜16 h. After this time, solvent was removed under reducedpressure, and the residue re-precipitated from MeOH:EtOAc to give Cpd.WW as a pale yellow amorphous solid (0.27 g, 91%), mp 287-292° C.(tar-liquid); ¹H NMR: 14.6 (v br s, 1H, quinolinyl-N⁺H), 13.84 (v br s,1H, pyridinyl-N⁺—H, 1H), 11.02 (s, 1H, ArNHAr), 10.95 (s, 1H, ArNHAr),8.80 (d, J=8.49 Hz, 1H, ArH), 8.58 (d, J=6.95 Hz, 1H, ArH), 8.28 (d,J=7.28 Hz, 2H, ArH), 8.06 (m, 2H, ArH), 7.79 (ddd, J=8.31, 6.80, 1.30Hz, 1H, ArH), 7.49 (d, J=8.50 Hz, 1H, ArH), 7.40 (d, J=8.53 Hz, 1H,ArH), 7.36 (d, J=8.78 Hz, 1H, ArH), 7.31 (m, 4H), 7.13 (d, J=7.10 Hz,2H, ArH), 6.76 (d, J=6.94 Hz, 1H, ArH), 3.44 (s, 3H, ArC(O)N(CH₃)Ar);HRMS (FAB⁺): calc. for C₂₈H₂₄N₅O (MH⁺) m/z 446.1981, found 446.1985;HPLC: 96.3%.

Example XX Preparation ofN-{4-[(2-amino-6-methyl-4-pyrimidinyl)amino]phenyl}-5-(4-quinolinylamino)-2-pyridinecarboxamidedihydrochloride (Cpd. XX)

N-[4-[(2-amino-6-methyl-4-pyrimidinyl)amino]phenyl]acetamidehydrochloride (16): To a mixture of 4-aminoacetanilide 14 (3.55 g, 23.64mmol) and 2-amino-4-chloro-6-methylpyrimidine 15 (3.73 g, 26 mmol) inethanol (50 mL) was added. C. HCl (2 drops). The reaction mixture wasstirred under reflux conditions for 2 h, cooled to 20° C. and theproduct was filtered washed with more ethanol and dried to giveessentially pure 16 (6.38 g, 92%); mp (EtOH) 203-207° C.; ¹H NMR[(CD₃)₂SO] δ 12.60 (br, 1H, N⁺H), 10.50 (br 1H, NH), 10.02 (s, 1H, NH),7.60-7.58 (br m, 6H, NH₂ & ArH), 6.13 (s, 1H, H-5″), 2.26 (s, 3H, CH₃),2.04 (s, 3H, CH₃); HRMS (FAB⁺), calc. for C₁₃H₁₆N₅O (M⁺¹) m/z 258.1355,found 258.1346.

N⁴-(4-aminophenyl)-6-methyl-2,4-pyrimidinediamine hydrochloride (17): Toa suspension of 16 (6.0 g, 20 mmol) was added 2N HCl (40 mL) and themixture was refluxed for 20 h. The solvents were evaporated to dryness,residue boiled in MeOH and diluted with EtOAc. The resulting precipitatewas filtered and washed with EtOAc and dried to give essentially pure 17(5.0 g, 97%); mp (MeOH/EtOAc) 275-280° C.; ¹H NMR [(CD₃)₂SO] δ 12.50(br, 1H, N⁺H), 10.75 (br s, 1H, NH), 9.75 (br s, 2H, NH₂), 7.85 (br s,4H, NH₂ & ArH)), 7.34 (d, J=8.6 Hz, 2H, ArH), 6.24 (br s, 1H, ArH), 2.79(s, 3H, CH₃), HRMS (FAB⁺) calc. for C₁₁H₁₄N₅ (M⁺¹) m/z 216.1249, found216.1247.

N-{4-[(2-amino-6-methyl-4-pyrimidinyl)amino]phenyl}-5-nitro-2-pyridinecarboxamide20. 5-Nitropyridine-2-carboxylic acid 18 (1.06 g, 6.31 mmol) wasrefluxed in POCl₃ (10 mL) for 1 h. (clear solution obtained), cooled to20° C. and excess POCl₃ was removed under vacuum. The resulting residuewas dissolved in 1,4-dioxane ((20 mL) and added slowly to a suspensionof 17 (1.44 g, 5.72 mmol) and N,N-diethylaniline (2.0 mL, 12.62 mmol) in1,4-dioane (20 mL). The reaction mixture was stirred at 20° C. for 3days. The resulting white precipitate was filtered and washed with more1,4-dioxane. The solid was stirred in aqueous NH₃ (20 mL) and theresulting red precipitate was filtered, washed with water andrecrystallized from MeOH to give 20 (708 mg, 31%); mp (MeOH)>290° C.; ¹HNMR ([(CD₃)₂SO] δ 10.74 (1H, NH), 9.43 (dd, J=2.6, 0.5 Hz, 1H, H-6),8.96 (s, 1H, NH), 8.81 (dd, J=8.6, 2.6 Hz, 1H, H-4), 8.38 (dd, J=8.6,0.6 Hz, 1H, H-3), 7.81 (d, J=9.0 Hz, 2H, H-2′ H-6′), 7.70 (d, J=9.1 Hz,2H, H-3′, 5′), 6.09 (s, 2H, NH₂), 5.87 (s, 1H, H-5″), 2.09 (s, 3H, CH₃);HRMS (FAB⁺) calc. for C₁₇H₁₆N₇O₃ (M⁺¹) m/z 366.1315, found 366.1314;Anal. calc. for C₁₇H₁₅N₇O₃.0.25 MeOH: C, 55.5; H, 4.4; N, 26.3; found,C, 55.7; H, 4.5; N, 26.2%.

5-amino-N-{4-[(2-amino-6-methyl-4-pyrimidinyl)amino]phenyl}-2-pyridinecarboxamide(21): To a suspension of 20 (523 mg, 1.43 mmol) was suspended in 1:1MeOH/THF (100 ml) was added 10% Pd/C (100 mg) and hydrogenated at 55 Hgmm. for 5 h. The reaction mixture was filtered and evaporated to drynessand recrystallized from DCM/Pet.ether to give 21 (649 mg, 96%); mp(DCM/Pet.ether)>290° C.; ¹H NMR [(CD₃)₂SO] δ 10.05 (s, 1H, NH), 8.85 (s,1H, NH), 8.25 (d, J=2.5 Hz, 1H, H-6), 7.82 (d, J=8.6 Hz, 1H, H-3), 7.73(d, J=9.0 Hz, 2H, H-2′, 6′), 7.60 (d, J=9.0 Hz, 2H, H-3′, 5′), 7.03 (dd,J=8.5, 2.7 Hz, 1H, H-4), 6.04 (brs, 2H, NH₂), 6.03 (brs, 2H, NH₂), 5.85(s, 1 H, H-5″), 2.08 (s, 3H, CH₃); Anal. calc. for C₁₇H₁₇N₇). 0.25H₂OC,60.1; H, 5.2; N, 28.9; found, C, 60.0; H, 5.2; N, 28.7%.

N-{4-[(2-amino-6-methyl-4-pyrimidinyl)amino]phenyl}-5-(4-quinolinylamino)-2-pyridinecarboxamidedihydrochloride (22) (Cpd. XX). To a solution of 21 (270 mg, 0.81 mmol)in EtOH (30 mL) and H₂O (15 mL) was added few drops of c.HCl, followedby 4-chloroquinoline (264 mg, 1.62 mmol, 2 eq) and stirred at 20° C.until dissolved. The reaction mixture was refluxed for 2 h, then more4-chloroquinoline (264 mg, 1.62 mmol) was added and refluxed for 20 h.The reaction mixture was diluted with EtOAc, boiled and cooled to 20° C.The resulting precipitate was filtered to give a pale yellow solid (95%clean by HPLC). This solid was stirred in aqueous NH₃. The resultingprecipitate was filtered washed with water, dried and recrystallizedfrom MeOH to give freebase of the product (300 mg). (This was 98% cleanby HPLC.) Then the free base was converted to HCl salt by adding 1.25MHCl in MeOH (2.5 mL), stirred 30 min, and evaporated to dryness. Theresidue was recrystallized from MeOH/EtOAc to give 22 (Cpd. XX) (297 mg69%); HPLC 99.2%; mp (MeOH/EtOAc)>290° C.; ¹H NMR [(CD₃)₂SO] δ 15.00(br, 1H, N⁺H), 12.50 (br, 1H, N⁺H), 11.23 (brs, 1H, NH), 10.74 (s, 1H,NH), 10.65 (brs, 1H, NH), 8.92 (d, J=2.3 Hz, 1H, ArH), 8.89 (d, J=8.5Hz, 1H, ArH), 8.66 (d, J=6.8 Hz, 1H, ArH), 8.31 (d, J=8.5 Hz, 1H, ArH),8.22 (dd, J=8.4, 2.5 Hz, 1H, ArH), 8.15 (d, J=7.9 Hz, 1H, ArH), 8.08 (t,J=7.9 Hz, 1H, ArH), 7.96 (d, 0.1=8.9 Hz, 2H, ArH), 7.87 (t, J=7.6 Hz,1H, ArH), 7.77 (br, 3H, ArH & NH₂), 7.13 (d, J=6.8 Hz, 1H, ArH), 6.18(s, 1H, ArH), 2.28 (s, 3H, CH₃) one of the aromatic CH signals was notobserved; Anal. calc. for C₂₆H₂₄Cl₂N₈O.0.5 H₂O: C, 57.4; H, 4.6; N,20.6; Cl, 13.0; found, C, 57.3; H, 4.8; N, 20.7; Cl, 12.7%.

Example YY Preparation ofN-{6-[(2-amino-6-methyl-4-pyrimidinyl)amino]-3-pyridinyl}-4-(4-quinolinylamino)benzamidedihydrochloride (Cpd. YY)

6-Methyl-M-(5-nitro-2-pyridinyl)-2,4-pyrimidinediamine (9). To asolution of 2-Amino-5-nitropyridine 1 (1.23 g, 8.84 mmol) and2-amino-4-chloro-6-methylpyrimidine (1.40 g, 9.72 mmol) in ethanol (30mL) was added few drops of c. HCl. The reaction mixture was refluxed for2 days, cooled to 20° C. and ethanol was evaporated to dryness. Theresulting brown glue was stirred in MeOH to obtain a filterableprecipitate. This was filtered and washed with more MeOH give theproduct as the hydrochloride salt. The ¹H NMR showed this was notcompletely clean. This material was converted to free base by stirringin aqueous NH₃, filtered and recrystallized to give 9 (682 mg, 31%); mp(MeO)>300° C.; ¹H NMR [(CD₃)₂SO] δ 10.47 (s, 1H, NH), 9.10 (dd, J=2.8,0.4 Hz, 1H, H-6′), 8.39 (dd, J=9.4, 2.8 Hz, 1H, H-4′), 8.28 (dd, J=9.3,0.3 Hz, 1H, H-3′), 6.63 (s, 1H, H-5), 6.39 (s, 2H, NH₂), 2.18 (s, 3H,CH₃); HRMS (EI⁺) calc. for C₁₀H₁₀N₆O₂(M⁺) m/z 246.0865, found 246.0866;Anal. calc. for C₁₀H₁₀N₆O₂: C, 48.8; H, 4.1; N, 34.1; found C, 48.7; H,4.2; N, 34.1%.

N⁴-(5-amino-2-pyridinyl)-6-methyl-2,4-pyrimidinediamine (10). Compound 9(634 mg, 246 mmol) was hydrogenated in MeOH (50 mL) with 10% Pd/C (100mg) at 45 Hg mm. for 20 h. The reaction mixture was filtered andevaporated to dryness to give 10 (550 mg, 99%); mp (MeOH) 230-233° C.;¹H NMR [(CD₃)₂SO] δ 8.96 (s, 1H, NH), 7.70 (d, J=8.7 Hz, 1H, H-3′), 7.65(d, J=2.5 Hz, 1H, H-6′), 6.95 (dd, J=8.8, 2.9 Hz, H-4′), 6.30 (s, 1H,H-5), 5.94 (s, 2H, NH₂), 4.84 (s, 2H, NH₂), 2.07 (s, 3 H, CH₃); HRMS(FAB⁺) calc. for C₁₀H₁₃N₆ (M⁺¹) m/z 217.1202, found 217.1202; Anal.calc. for C₁₀H₁₂N₆; C, 55.5; H, 5.6; N, 38.9; found C, 55.3; H, 5.7; N,38.6%.

N-{6-[(2-amino-6-methyl-4-pyrimidinyl)amino]-3-pyridinyl}-4-nitrobenzamide(11). To a suspension of 10 (500 mg, 2.31 mmol) and N,N-diethylaniline(1 ml, 1.5 eq) in 1,4-dioxane (20 ml) at 0° C. was added dropwise asolution of p-nitrobenzoylchloride (429 mg, 12.31 mmol). The reactionmixture was stirred at 20° C. for 2 h. TLC and mass spectrum showedstill presence of 10. Therefore more p-nitrobenzoylchloride (43 mg, 0.1eq) was added and stirred for 20 h. The resulting precipitate wasfiltered and washed with more 1,4-dioxane. The collected solid wasstirred in aqueous NH₃, filtered washed with water and dried to giveessentially pure 11 (821 mg, 97%); mp (MeOH)>300° C.; ¹H NMR [(CD₃)₂SO]δ 10.58 (s, 1H, NH), 9.56 (s, 1H, NH), 8.65 (d, J=2.5 Hz, 1H, H-2′),8.38 (d, J=8.9 Hz, 2H, H-3, 5), 8.20 (d, J=9.2 Hz, 2H, H-2, 6), 8.17 (d,J=9.1 Hz, 1H, H-5′), 8.02 (dd, J=9.0, 2.6 Hz, 1 H, H-4′), 6.45 (s, 1H,H-5″), 6.15 (s, 2H, NH₂), 2.12 (s, 3H, CH₃); HRMS (FAB⁺) (M⁺¹) m/z calc.for C₁₇H₁₆N₇O₃(M⁺¹) m/z, 366.1315, found 366. 1314; Anal. calc. forC₁₇H₁₅N₇O₃: C, 55.9; H, 4.1; N, 26.8, found C, 55.6; H, 4.2; N, 26.8%.

4-amino-N-{6-[(2-amino-6-methyl-4-pyrimidinyl)amino]-3-pyridinyl}benzamide12. To a suspension of 11 (790 mg, 2.16 mmol) in 1:1 MeOH/THF (100 ml)was added 10% Pd/C (100 mg) and hydrogenated at 45 Hg mm. for 20 h. Thereaction mixture was filtered and evaporated to dryness andrecrystallized from DCM/Pert.ether to give 12 (695 mg, 96%); mp(DCM/pet.ether)>300° C.; ¹H NMR [(CD₃)₂SO] δ 9.78 (s, 1H, NH), 9.45 (s,1H, NH), 8.63 (dd, J=2.4, 0.3 Hz, 1H, H-2′), 8.09 (d, J=9.0 Hz, 1H,H-5′), 7.98 (dd, J=9.0, 2.6 Hz, 1H, H-4′), 7.72 (d, J=8.7 Hz, 2H, H-2,6), 6.61 (d, J=8.7 Hz, 2H, H-3, 5), 6.43 (s, 1H, H-5″), 6.13 (s, 2H,NH₂), 5.73 (s, 2H, NH₂), 2.12 (s, 3H, CH₃); HRMS (FAB⁺) calc. forC₁₇H₁₈N₇O (M⁺¹) m/z 336.1573, found 336.1578; Anal. calc. forC₁₇H₁₇N₇O.0.25H₂O: C, 60.1; H, 5.2; N, 28.9; C, 60.2; N, 5.3; N, 29.0%.

N-{6-[(2-amino-6-methyl-4-pyrimidinyl)amino]-3-pyridinyl}-4-(4-quinolinylamino)benzamidedihydrochloride (13) (Cpd. YY). To a solution of 12 (250 mg, 0.75 mmol)in EtOH (40 mL) and H₂O (20 mL) was added few drops of c.HCl, followedby 4-chloroquinoline (159 mg, 0.98 mmol, 1.3 eq) and stirred at 20° C.until dissolved. The reaction mixture was refluxed for 4 h, then more4-chloroquinoline (100 mg) was added and refluxed for 20 h. The reactionmixture was diluted with EtOAc, boiled and cooled to 20° C. Theresulting precipitate was filtered to give a pale yellow solid. (75%clean by HPLC) This solid was stirred in aqueous NH₃. The resultingprecipitate was filtered washed with water and dried to give freebase ofthe product. This was 96% clean by HPLC. Then the free base wasconverted to HCl salt by adding 1.25M HCl in MeOH (2.5 mL), stirred 30min, and evaporated to dryness. The residue was stirred in MeOH (10 mL),filtered and dried to give 13 (Cpd. YY) (365 mg 91%); HPLC 98.9%; mp(MeOH)>290° C.; ¹H NMR [(CD₃)₂SO] δ 14.70 (br 1H, N⁺H), 12.98 (br, 1H,N⁺H), 11.12 (s, 1H, NH), 11.01 (s, 1H, NH), 10.68 (s, 1H, NH), 8.88 (d,J=3.0 Hz, 1H, ArH), 8.84 (d, J=8.6 Hz, 1H, ArH), 8.62 (d, J=6.9 Hz, 1H,ArH), 8.26 (dd, J=9.0, 2.6 Hz, 1H, ArH), 8.22 (d, J=8.6 Hz, 2H, ArH),8.15-8.05 (m, 2H, ArH), 7.86 (ddd, J=7.6, 6.8, 1.5 Hz, 1H, ArH), 7.70(d, J=8.6 Hz, 2H, ArH), 7.01 (d, J=6.9 Hz, 1H, ArH), 7.02 (v.br, 1H,ArH), 2.32 (s, 3H, CH₃), the signals for NH₂ and one of aromatics werenot observed; HRMS (FAB⁺) calc. for C₂₆H₂₃N₈O (M⁺¹) m/z 463.1995, found463.1996; Anal. calc for C₂₆H₂₄N₈Cl₂O.HCl.0.25H₂O: C, 54.2; H, 4.5; N,19.4; Cl, 18.5; found C, 54.2; H, 4.5; N, 19.3; Cl, 17.7%.

Example ZZ Preparation ofN-{5-[(2-amino-6-methyl-4-pyrimidinyl)amino]-2-pyridinyl}-4-(4-quinolinylamino)benzamidedihydrochloride (Cpd. ZZ)

N-{5-[(2-amino-6-methyl-4-pyrimidinyl)amino]-2-pyridinyl}-acetamide (4).To a solution of N-(5-amino-2-pyridinyl)acetamide 2 (1.04 g, 6.88 mmol),and 2-amino-4-chloro-6-methylpyrimidine (1.09 g, 7.57 mmol) in EtOH (30mL) was added 2 drops c. HCl. The reaction mixture was refluxed for 2 h,cooled to 20° C. The resulting precipitate was filtered washed with moreethanol and dried to give (1.85 g,) further material (165 mg) wasisolated from mother liquor concentration. Total yield of 4 99.3% mp(MeOH/EtOAc)>300° C. ¹H NMR [(CD₃)₂SO] δ 12.77 (br, 1H, NH), 10.72 (br,1H, NH), 10.44 (s, 1H, NH), 8.63 (br s, 1H, ArH), 8.13-8.06 (m, 2H,ArH), 7.79 (v. br, 2H, NH₂), 6.18 (s, 1H, ArH), 2.30 (s, 3H, COCH₃),2.09 (s, 3H, CH₃); HRMS (FAB⁺) calc. for C₁₂H₁₅N₆O (M⁺¹) m/z 259.1307,found 259.1304; Anal. Calc. for C₁₂H_(15Cl)N₆O.H₂O: C, 46.1; H, 5.5; N,26.9; Cl, 11.3; found C, 45.9; H, 5.5; N, 26.4; Cl, 11.45%.

N⁴-(6-amino-3-pyridinyl)-6-methyl-2,4-pyrimidinediamine (5). Asuspension of 4 (1.53 g, 5.19 mmol), in 1,4-dioxane/MeOH (1:1, 100 mL)and 2 N HCl [10 mL, H₂O mL+c. HCl 2 mL)] was refluxed 24 h. The solventswere evaporated to dryness and the residue was basified with aq NH₃. Theresulting solution was extracted with EtOAc (10×50 mL), dried (Na₂SO₄)and evaporation of the solvent gave 5 (1.11 g, 99%). A small sample wasrecrystallized from DCM/Pet.ether; mp (DCM/Pet.ether) 183-186° C.; ¹HNMR [(CD₃)₂SO] δ 8.41 (s, 1H, NH), 8.01 (d, J=2.5 Hz, 1H, H-2′), 7.56(dd, J=8.7, ′), 6.42 (d, □□□2.6 Hz, 1H, H-4 J=8.8 Hz, 1H, H-5′), 5.90(s, 2H, NH₂), 5.67 (s, 1H, H-5), 5.59 (s, 2 H, NH₂H, CH□□), 2.03 (s, 3₃)HRMS (EI⁺) calc. for C₁₀H₁₂N₆ (M⁺) m/z 216.1123, found 216.1124; Anal.Calc. for C₁₀H₁₂N₆.0.25H₂O: C, 54.4; H, 5.7; N, 38.1; found, C, 54.4; H,5.7; N, 37.9%.

N-{5-[(2-amino-6-methyl-4-pyrimidinyl)amino]-2-pyridinyl}-4-nitrobenzamide)(6). To a suspension of 5 (830 mg, 3.84 mmol) and N,N-diethylaniline(1.0 mL, 5.76 mmol) in 1,4-dioxane (20 mL) at 0° C. was added dropwise asolution of 4-nitrobenzoylchloride (720 mg, 3.88 mmol) in 1,4-dioxane(20 mL). After the addition was completed the reaction mixture wasstirred at 20° C. for 1 h, then dioxane was removed under vacuum. Theresidue was stirred in H₂O (50 mL) and the resulting precipitate wasfiltered washed with aqueous NH₃, H₂O, and pet.ether. The residue wasboiled in MeOH and the insoluble red solid was collected. This processwas repeated 3 more times to give essentially pure 6 (903 mg, 65%), [ifrepeating the product should be filtered off from dioxane to avoid extrasteps]; mp (MeOH) 294-297° C.; ¹H NMR [(CD₃)₂SO] 10.99 (s, 1H, NH), 9.14(s, 1H, NH), 8.68 (d, J=2.5 Hz, 1H, H-6′), 8.32 (d, J=8.9 Hz, 2H,H-3&5), 8.27 (dd, J=9.0, 2.7 Hz, 1H, H-4′), 8.23 (d, J=8.9 Hz, 2H,H-2&H-6), 8.08 (d, J=9.0 Hz, 1H, H-3′), 6.18 (s, 2H, NH₂), 5.88 (s, 1H,H-5″), 2.11 (s, 3H, CH₃); HRMS (FAB⁺), calc. for C₁₇H₁₆N₇O₃ (M⁺¹) m/z366.1315, found 366.1312; Anal. calc. for C₁₇H₁₅N₇O₃.CH₃OH: C, 54.4; H,4.8; N, 24.7; found C, 54.5; H, 4.8; N, 24.7%.

4-amino-N-{5-[(2-amino-6-methyl-4-pyrimidinyl)amino]-2-pyridinyl}benzamide(7). To a suspension of 6 (802 mg, 2.19 mmol) in 1:1 MeOH/THF (100 ml)was added 10% Pd/C (100 mg) and hydrogenated at 45 Hg mm. for 20 h. Thereaction mixture was filtered and evaporated to dryness andrecrystallized from DCM/Pert.ether to give 7 (697 mg, 95%); mp(DCM/pet.ether) 157-161° C.; ¹H NMR [(CD₃)₂SO] δ 9.98 (s, 1H, NH), 9.03(s, 1H, ⁻NH), 8.59 (d, J=2.4 Hz, 1H, H-6′), 8.16 (dd, J=9.0, 2.7 Hz, 1H,H-4′), 8.04 (d, J=8.9 Hz, 1H, H-3′), 7.77 (d, J=8.7 Hz, 2H, H-2, 6),6.57 (d, J=8.7 Hz, 2H, H-3, 5), 6.13 (s, 2H, NH₂), 5.13 (s, 1H, H-5″),5.74 (s, 2H, NH₂), 2.10 (s, 3H, CH₃); HRMS (FAB⁺) calc. for C₁₇H₁₈N₇O(M⁺¹) m/z 336.1573, found 366.1574; Anal. calc. for C₁₇H₁₇N₇O.2H₂O: C,55.0; H, 5.7; N, 26.4; found C, 55.2; H, 5.7; N, 26.3%.

N-{5-[(2-amino-6-methyl-4-pyrimidinyl)amino]-2-pyridinyl}-4-(4-quinolinylamino)benzamidedihydrochloride (8) (Cpd. ZZ). To a solution of 7 (241 mg, 0.72 mmol) inEtOH (20 mL) and H₂O (10 mL) was added 4-chloroquinoline (153 mg, 0.94mmol, 1.3 eq) and stirred at 20° C. until dissolved. Then c.HCl fewdrops were added and refluxed for 24 h. The reaction mixture was dilutedwith EtOAc, boiled and cooled to 20° C. The resulting precipitate wasfiltered to give a pale yellow solid, this was recrystallized fromMeOH/EtOAc to give 350 mg of the product this was 84% clean by HPLC.This solid was stirred in aqueous NH₃ to convert to free base of theproduct, resulting precipitate was filtered washed with water and driedto give 204 mg. This was 98% clean by HPLC. The free base was convertedto HCl salt by adding 1.25M HCl in MeOH (1 mL), stirred 30 min, filteredand dried to give 8 (Cpd. ZZ) (224 mg 58%); HPLC 100%; mp (MeOH)>295°C.; ¹H NMR [(CD₃)₂SO] δ 14.75 (br, 1H, N⁺H), 12.89 (bs, 1H, N⁺H), 11.14(s, 1H, NH), 10.99 (bs, 1H, NH), 10.94 (s, 1H, NH), 8.87 (d, J=8.5 Hz,1H, ArH), 8.77 (br 1H, ArH), 8.61 (d, J=6.9 Hz, 1H, ArH), 8.31 (br, 1H,ArH), 8.23 (d, J=8.6 Hz, 2H, ArH), 8.13 (dd, J=8.0, 0.9 Hz, 1H, ArH),8.07 (td, J=7.7, 0.9 Hz, 1H, ArH), 7.85 (td, J=7.7, 1.2 Hz, 1H, ArH),7.67 (d, J=8.6 Hz, 2H, ArH), 7.02 (d, J=6.9 Hz, 1H, ArH), 6.26 (s, 1H,ArH), 2.31 (s, 3H, CH₃); HRMS (FAB⁺), calc. for C₂₆H₂₃N₈O (M⁺¹) m/z4631995, found 463.1994; Anal. calc. for C₂₆H₂₄Cl₂N₈O.HCl. H₂O: C, %2.9;H, 4.6; N, 19.0; Cl, 18.0. Found C, 53.1; H, 4.6; N, 19.2; Cl, 17.9%.

Example AAA Preparation ofN-[4-(pyridin-4-ylthio)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. AAA)

4-(Pyridin-4-ylthio)aniline (3). To a solution of 4-aminobenzenethiol 1(5.10 g, 40.74 mmol) in dry DMF (90 mL) were sequentially added4-chloropyridine hydrochloride 2 (6.41 g, 42.68 mmol) and anhydrousK₂CO₃ (14.70 g, 106.34 mmol), and the resulting suspension was stirredvigorously at room temperature for ˜3 h. After this time, the reactionmixture was diluted with EtOAc (100 mL) and H₂O (100 mL), and theorganic layer separated. The aqueous layer was extracted further withEtOAc (100 mL×2), and the organic extracts were then combined, washedwith brine, and finally dried over anhydrous MgSO₄. Solvent was removedunder reduced pressure, and washing of the resulting residue with 1:1Et₂O:hexanes (300 mL) afforded amine 3 as fine grey-white crystallinesolid (4.64 g, 56%), mp (MeOH:EtOAc) 171-173° C.; ¹H NMR [(CD₃)₂SO]: δ5.64 (s, 2H, NH₂), 6.67 (ddd, J=9.40, 4.78, 2.81 Hz, 2H, ArH), 6.90 (dd,J=4.61, 1.59 Hz, 2H, ArH), 7.20 (ddd, J=9.40, 4.78, 2.81 Hz, 2H, ArH),8.29 (dd, J=4.61, 1.59 Hz, 2H, ArH); HRMS: Calc. for C₁₁H₁₁N₂S (M⁺)203.0643, found 203.0641.

4-Nitro-N-[4-(pyridin-4-ylthio)phenyl]benzamide (5). To a solution ofamine 3 (2.03 g, 10.06 mmol) in dry dioxane (70 mL) were sequentiallyadded dry pyridine (4.05 mL, 50.28 mmol) and 4-nitrobenzoyl chloride 4(3.19 g, 17.18 mmol, as solution in 30 mL of dry dioxane), and theresulting mixture was stirred at −50° C. for 14 h. After this time, theresultant yellow solid was isolated by filtration, and washedsequentially with dioxane, EtOAc, and hexanes. The resulting solid wasre-dissolved in MeOH (˜5 L), and this solution was filtered throughCelite to remove undissolved impurities, and then concentrated to asmaller volume under reduced pressure. The resulting solid was collectedby filtration, then re-suspended in and washed sequentially with EtOH,MeOH, and EtOAc, and finally collected again by filtration. Theresulting material was washed with hexanes and dried under high-vacuumto afford amide 5 as an amorphous yellow powdery solid, mp 295-298° C.;¹H NMR [(CD₃)₂SO]: δ 7.38 (d, J=6.15 Hz, 2H, ArH), 7.55 (ddd, J=9.42,4.45, 2.59 Hz, 2H, ArH), 8.07 (m, 2H, ArH), 8.24 (ddd, J=9.21, 4.32,2.31 Hz, 2H, ArH), 8.39 (dd, J=6.92, 1.97 Hz, 2H, ArH), 8.53 (d, J=6.6Hz, 2H, ArH), 10.97 (s, 1H, —C(O)NH—); HRMS: Calc. for C₁₈H₁₄N₃O₃S (M⁺)352.0756, found 352.0755.

4-Amino-N-[(4-(pyridin-4-ylthio)phenyl]benzamide (6). To a refluxingsolution of nitro compound 5 (0.54 g, 1.53 mmol) in 2:1 EtOH:H₂O (100mL) were sequentially added Fe dust (0.54 g, 9.72 mmol) and c.HCl (2mL), and the resulting suspension was refluxed for 1 h. After this time,the hot reaction mixture was filtered through a pad of Celite, andsolvent was removed under reduced pressure. The residue was redissolvedin MeOH, and the resulting solution stirred with Celite overnight. Theresulting suspension was filtered through a pad of Celite, and thefiltrate acidified with 1.25 M methanolic HCl. The solution wasconcentrated to a smaller volume under reduced pressure, and theresulting solid removed by filtration through a pad of Celite. Thefiltrate was re-acidified and treated as before (twice), finallyaffording an amorphous ochre solid (0.38 g, 77%) which was used withoutfurther purification; ¹H NMR [(CD₃)₂SO]: δ 5.79 (s, 2H, —NH₂), 6.62 (d,J=8.58 Hz, 2H, ArH), 6.98 (d, J=5.96 Hz, 2H, ArH), 7.53 (d, J=8.61 Hz,2H, ArH), 7.74 (d, J=8.58 Hz, 2H, ArH), 7.96 (d, J=8.61 Hz, 2H, ArH),8.34 (d, J=5.35 Hz, 2H, ArH), 10.02 (s, 1H, —C(O)NH—); LCMS (APCI⁺): 322(100%).

N-[4-(pyridin-4-ylthio)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. AAA). To a solution of amine 5 (0.31 g, 0.97 mmol)in 20% aq. EtOH (100 mL) were sequentially added 4-chloroquinoline 7(0.33 g, 2.02 mmol) and c.HCl (0.20 mL, 8.71 mmol), and the resultingsuspension refluxed for 15 h. After this time, solvent was removed underreduced pressure, and the residue dried via two MeOH azeotrope cycles.The resulting solid was purified by column chromatography (twice) onsilica gel, eluting with 5%→10%→20% MeOH:CH₂Cl₂, to afford a solidresidue which was re-precipitated from MeOH:methanolic HCl:EtOAc to giveCpd. AAA as an amorphous yellow solid (0.15 g, 29%), mp (EtOAc:MeOH)306-310° C.; ¹H NMR [(CD₃)₂SO]: δ 7.01 (d, J=6.94 Hz, 1H, ArH), 7.45 (d,J=6.77 Hz, 2H, ArH), 7.70 (m, 4H), 7.86 (m, 1H), 8.11 (m, 4H), 8.22 (dd,J=6.82, 1.79 Hz, 2H, ArH), 8.55 (d, J=6.77 Hz, 2H, ArH), 8.62 (d, J=6.94Hz, 1H, ArH), 8.89 (d, J=8.35 Hz, 1H, ArH), 10.78 (s, 1H, ArNHAr), 11.19(s, 1H, —C(O)NH—), 14.72 (br s, 1H, pyridinium-N⁺—H) [quinolinium N⁺—Hnot visible]; HRMS: Calc. for C₂₇H₂₁N₄OS (M⁺) 449.1436, found 449.1441;HPLC: 99.3%.

Example BBB Preparation ofN-[4-(2-aminopyridin-4-ylthio)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. BBB)

4-(4-Aminophenylthio)pyridin-2-amine (9). To a solution of4-aminobenzene thiol 1 (9.49 g, 75.77 mmol) in dry DMF (54 mL) weresequentially added 4-chloro-2-aminopyridine 8 (3.70 g, 28.80 mmol) anddry K₂CO₃ (10.70 g, 107.97 mmol), and the resulting yellow suspensionstirred at ˜120° C. (bath temperature) for ˜45 min. After this time, theresultant brown-black suspension was cooled to room temperature, andthen diluted with H₂O and EtOAc. The resulting mixture was extractedwith EtOAc (×2), and the organic fractions were combined and washed withbrine, and then dried over MgSO₄. Solvent was removed under reducedpressure, and the residue was then re-dissolved in a small amount ofMeOH, and filtered though a pad of silica gel. Solvent was removed underreduced pressure to afford amine 9 (5.66 g, 90%) as an amorphouscreamy-purple solid, mp: 141-143° C.; ¹H NMR [(CD₃)₂SO]: δ 5.56 (br s,2H, NH₂), 5.76 (br s, 2H, NH₂), 5.94 (d, J=1.22 Hz, 1H, ArH), 6.10 (dd,J=5.46, 1.68 Hz, 1H, ArH), 6.64 (ddd, J=9.37, 4.78, 2.79 Hz, 2H, ArH),7.17 (ddd, J=9.38, 4.74, 2.79 Hz, 2H, ArH), 7.65 (d, J=5.44 Hz, 1H,ArH); HRMS: Calc. for C₁₁H₁₂N₃S (MH⁺) m/z 218.0753, found 218.0750.

N-[4-(2-Aminopyridin-4-ylthio)phenyl]-4-nitrobenzamide (10). To asolution of amine 9 (1.09 g, 5.01 mmol) in dry dioxane (30 mL) weresequentially added dry pyridine (2.08 mL, 25.04 mmol) and 4-nitrobenzoylchloride 4 (1.60 g, 8.60 mmol; added as a solution in 20 mL of dry DMF),and the resulting mixture was stirred at 55-60° C. (bath temperature)for ˜4 h. After this time, the resultant solid was collected byfiltration, and washed sequentially with dioxane, EtOAc, and hexanes.The crude product was re-precipitated from MeOH:methanolic HCl:EtOAc toafford nitro compound 10 as an amorphous yellow solid (1.19 g, 64%),mp>300° C.; ¹H NMR [(CD₃)₂SO]: δ 13.35 (br s, 1H, quinoline-N⁺—H), 10.97(s, 1H, ArC(O)NHAr), 8.39 (ddd, J=9.25, 4.40, 2.37 Hz, 2H, ArH), 8.23(ddd, J=9.20, 4.34, 2.31 HZ, 2H, ArH), 8.06 (ddd, J=9.43, 4.53, 2.62 Hz,2H, ArH), 7.81 (m, 3H, ArH & ArNH₂), 7.66 (ddd, J=9.41, 4.52, 2.61 Hz,2H, ArH), 6.65 (dd, J=6.87, 1.91 Hz, 1H, ArH), 6.29 (d, J=1.73 Hz, 1H,ArH); HRMS: Calc. for C₁₈H₁₅N₄O₃S (MH⁺) m/z 367.0865, found 367.0865.

4-Amino-N-[4-(2-aminopyridin-4-ylthio)phenyl]benzamide hydrochloride(11). Nitro compound 10 (0.83 g, 2.07 mmol) was suspended in 2:1EtOH:H₂O (100 mL) and the resulting suspension brought to reflux. Tothis mixture was sequentially added Fe dust (0.54 g, 9.59 mmol) andc.HCl (2 mL), and the resulting dark orange suspension was refluxed for1 h. After this time, the resulting yellow suspension was filtered hotthrough a pad of Celite, and the solvent removed under reduced pressure.The residue was re-suspended on H₂O, to which was added a quantity ofCelite, and the resulting suspension was stirred overnight. After thistime, the suspension was filtered through a pad of Celite, and solventwas removed under reduced pressure to afford crude amine 11 as anamorphous off-white solid (0.65 g, 93%), which was used without furtherpurification. ¹H NMR [(CD₃)₂SO]: δ 9.98 (s, 1H, ArC(O)NHAr), 7.91 (ddd,J=9.40, 4.48, 2.58 Hz, 2H, ArH), 7.72 (m, 3H, ArH), 7.48 (ddd, J=9.37,4.39, 2.55 Hz, 2H, ArH), 6.61 (d, J=7.85 Hz, 2H, ArH), 6.18 (dd, J=5.46,1.68 Hz, 1H, ArH), 5.82 (d, J=1.36 Hz, 1H, ArH), 6.01 (br s, 2H, ArNH₂),5.82 (br s, 2H, ArNH₂); HRMS: Calc. for C₁₈H₁₁N₄OS (MH⁺) m/z 337.1123,found 337.1126.

N-[4-(2-aminopyridin-4-ylthio)phenyl]-4-(quinolin-4-ylamino)-benzamidehydrochloride (Cpd. BBB). 4-Chloroquinoline 7 (0.66 g, 4.01 mmol) andc.HCl (0.52 mL, 17.13 mmol) were sequentially added to a solution ofamine 11 (0.63 g, 1.88 mmol) in 20% aq. EtOH (100 mL), and the resultingmixture refluxed for 3 h. After this time, solvent was removed underreduced pressure, and the residue was dried via two MeOH azeotropecycles. The residue was then re-precipitated twice from MeOH:methanolicHCl:EtOAc to give Cpd. BBB (0.55 g, 54%) as an amorphous yellow solid,mp 225-239° C.; ¹H NMR [(CD₃)₂SO]: δ 14.10 (v v br s, 2H, quinolinyl-N⁺H& pyridinyl-N⁺H), 11.14 (s, 1H, ArNHAr), 10.75 (s, 1H, ArC(O)NHAr), 8.87(d, J=8.48 Hz, 1H, ArH), 8.62 (d, J=6.92 Hz, 1H, ArH), 8.20 (d, J=8.61Hz, 2H, ArH), 8.09 (m, 4H, ArH), 7.82 (m, 4H, ArH & ArNH₂), 7.68 (m, 4H,ArH), 7.01 (d, J=6.91 Hz, 1H, ArH), 6.66 (dd, J=6.88, 1.87 Hz, 1H, ArH),6.31 (d, J=1.74 Hz, 1H, ArH); HRMS: Calc. for C₂₇H₂₂N₅OS (MH⁺) m/z464.1541, found 464.1541; HPLC: 97.4%.

Example CCC Preparation ofN-[4-(2-hydroxypyridin-4-ylthio)phenyl]-4-(quinolin-4-ylamino)benzamidehydrochloride (Cpd. CCC)

4-(4-Aminophenylthio)pyridin-2-ol (13). To a solution of 4-aminobenzenethiol 1 (3.68 g, 29.40 mmol) in dry DMF (54 mL) were sequentially added4-chloro-2-hydroxypyridine 12 (0.49 g, 3.81 mmol) and dry K₂CO₃ (10.70g, 107.97 mmol), and the resulting yellow suspension stirred at −120° C.(bath temperature) for ˜1 h. LCMS analysis of the reaction mixture afterthis time showed that there was still much 12 present, thus a furtherquantity of 1 (1.28 g, 10.22 mmol) was added (as a solution in 10 mL ofdry DMF). After 1 h, LCMS and TLC analysis showed the reaction to becomplete, so the reaction mixture was cooled to room temperature,diluted with H₂O, and extracted with EtOAc (×3). The combined organicextracts were washed with brine, dried over anhydrous MgSO₄, and solventwas removed under reduced pressure. The reside was purified by columnchromatography on silica gel, eluting with 1%→10% MeOH:CH₂Cl₂, to affordamine 13 as an amorphous off-white solid (0.47 g, 56%), which was usedwithout further purification; ¹H NMR [(CD₃)₂SO]: δ 11.20 (br s, 1H,ArOH), 7.18 (m, 3H, ArH), 6.66 (ddd, J=9.38, 4.76, 2.80 Hz, 2H, ArH),5.89 (dd, J=6.95, 1.92 Hz), 5.64 (br s, 2H, ArNH₂), 5.55 (d, J=1.79 Hz,1H, ArH); HRMS: Calc. for C₁₁H₁₁N₂OS m/z 219.0592, found 219.0591.

N-[4-(2-hydroxypyridin-4-ylthio)phenyl]-4-nitrobenzamide (14). To asolution of amine 13 (0.47 g, 2.13 mmol) in dry dioxane (120 mL) weresequentially added dry pyridine (0.86 mL, 10.65 mmol) and 4-nitrobenzoylchloride 4 (0.70 g, 3.76 mmol, added as solution in 20 mL of dry DMF),and the resulting mixture was stirred at ˜50° C. (bath temperature)overnight. After this time, the reaction mixture was cooled to roomtemperature, and then combined with a quantity of silica. Solvent wasremoved under reduced pressure, and the resulting silica adsorbate waspurified by column chromatography on silica gel, eluting with 1%→20%MeOH:CH₂Cl₂ (with 0.5% aq.NH₃), to afford cleaner 14. This material waswashed with MeOH, and the undissolved solid filtered off and dried, toafford a first batch of nitro compound 14 (0.27 g). The filtrate wasconcentrated under reduced pressure, and the residue washed with a smallamount of MeOH, and then dried, affording a further quantity of 14 (0.30g, overall 68%), mp 255-260° C. (dark powder→tar); ¹H NMR [(CD₃)₂SO]: δ10.86 (s, 1H, ArC(O)NHAr), 8.38 (ddd, J=9.22, 4.34, 2.33 Hz, 2H ArH),8.22 (ddd, J=9.24, 4.32, 2.31 Hz, 2H, ArH), 7.98 (ddd, J=9.40, 4.48,2.57 Hz, 2H ArH), 7.61 (ddd, J=9.33, 4.46, 2.56 Hz, 2H, ArH), 7.38 (d,J=6.95 Hz, 1H, ArH), 6.05 (dd, J=6.94, 1.93 Hz, 1H, ArH), 5.73 (d,J=1.78 Hz, 1H, ArH) [—OH signal very broad around 5.6 ppm]; HRMS: Calc.for C₁₈H₁₄N₃O₄S (MH⁺) m/z 368.0705, found 368.0711.

4-Amino-N-[4-(2-hydroxypyridin-4-ylthio]phenyl)benzamide (15). Nitrocompound 14 (0.48 g, 1.18 mmol) was suspended in 2:1 EtOH:H₂O (100 mL)and the resulting suspension brought to reflux. To this mixture wassequentially added Fe dust (0.30 g, 9.59 mmol) and c.HCl (2 mL), and theresulting suspension refluxed for 15 min. TLC and LCMS analysis at thistime showed that the reaction was incomplete, thus further quantities ofFe dust (0.80 g, 14.29 mmol) and c.HCl (10 mL) were added, and themixture refluxed for a further 50 min. After this time, the reaction wascomplete, and thus the reaction mixture was filtered hot through a padof Celite, and solvent was removed under reduced pressure. The residuewas dried via two MeOH-azeotrope cycles, and then re-dissolved in MeOHand stirred overnight with Celite and activated charcoal. The resultingslurry was filtered through a pad of Celite, and solvent was removedunder reduced pressure. The residue was re-dissolved in MeOH andadsorbed onto a quantity of silica gel, and the resulting silicaadsorbate was purified by column chromatography on silica gel, elutingwith 1%→20% MeOH:CH₂Cl₂ (with 0.5% aq.NH₃), to afford crude 15. Thismaterial was purified further by re-precipitation from MeOH:methanolicHCl:EtOAc, to afford amine 15 as an amorphous cream solid (15 mg,3%—presumably much material lost by adsorption to activated charcoal;latter was extracted several times, to little avail); ¹H NMR [(CD₃)₂SO]:δ 11.40 (v v br s, 1H, quinolinyl-N⁺—H), 10.08 (s, 1H, ArH), 7.94 (ddd,J=9.40, 4.51, 2.59 Hz, 2H, ArH), 7.78 (d, J=8.67 Hz, 2H, ArH), 7.52(ddd, J=9.40, 4.49, 2.61 Hz, 2H, ArH), 7.27 (d, J=6.95 Hz, 1H, ArH),6.71 (d, J=8.52 Hz, 2H, ArH), 5.96 (dd, J=6.96, 1.93 Hz, 1H, ArH) [ArOH& ArNH₂ not visible]; LCMS (APCI⁺): 338 (100%), 423 (60%), 169 (40%).

N-[4-(2-Hydroxypyridin-4-ylthio)phenyl]-4-(quinolin-4-ylamino)-benzamidehydrochloride (Cpd. CCC). 4-Chloroquinoline 7 (12 mg, 0.07 mmol) andc.HCl (30 μl, 17.13 mmol) were sequentially added to a solution of amineI5 (13 mg, 0.032 mmol) in 20% aq. EtOH (6 mL), and the resulting mixturerefluxed for 18 h. After this time, solvent was removed under reducedpressure, and the residue was dried via two MeOH-azeotrope cycles. Theresulting material was re-dissolved in MeOH and adsorbed onto a quantityof silica gel, and the resulting silica adsorbate was purified by columnchromatography on silica gel, eluting with 1%→10% MeOH:CH₂Cl₂ (with 0.5%aq.NH₃), to afford cleaner material. This material was purified furtherby re-precipitation from MeOH:methanolic HCl:EtOAc, to afford Cpd. CCCas an amorphous yellow solid (7 mg, 41%), mp 209-214° C.; ¹H NMR[(CD₃)₂SO]: δ 14.42 (br s, 1H, 1H, quinolinyl-N⁺H), 11.34 (br s, 1H,pyridinyl-N⁺—H), 10.99 (s, 1H, ArNHAr), 10.61 (s, 1H, ArC(O)NHAr), 8.77(d, J=8.60 Hz, 1H, ArH), 8.62 (d, J=6.90 Hz, 1H, ArH), 8.19 (d, J=8.60Hz, 2H, ArH), 8.07 (m, 2H), 8.00 (dd, J=6.88, 1.83 Hz, 2H, ArH), 7.87(septet, J=11.29, 8.36, 5.40, 2.76 Hz, 1H, ArH), 7.69 (d, J=8.55 Hz, 2H,ArH), 7.60 (d, J=8.60 Hz, 2H, ArH), 7.27 (d, J=6.97 Hz, 1H, ArH), 7.06(d, J=6.94 Hz, 1H, ArH), 5.97 (d, J=6.96, 1.88 Hz), 1H, ArH), 5.63 (d,J=1.76 Hz, 1H, ArH) [ArOH not visible]; HRMS: Calc. for C₂₇H₂₁N₄O₂S(MH⁺) m/z 465.1385, found 465.1389; HPLC: 93.5%.

Unless specified otherwise, all solvents and reagents used in thefollowing Examples DDD, EEE, FFF and GGG below were available fromSigma-Aldrich (St. Louise, Mo.) or VWR International (West Chester, Pa.)and used as supplied or purified by standard laboratory methods asrequired. NMR spectra were recorded on a Varian Unity at 400 MHz (¹H) at25° C. Chemical shifts are reported in ppm and referenced internally toresidual CHCl₃ for (d 7.26) or CH₃OH for (d 3.33). Low resolution massspectrometry was performed by The Mass Spectrometry and Proteomics Corefacility at the University of Utah. Flash chromatography was performedon Combifalsh (Yamazen) with normal phase silica gel column (RediSep)and CH₇Cl₂/CH₃OH solvent system. TLC used pre-coated silica gel aluminumsheets.

Example DDD Preparation of4-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine-2-carboxylicacid (Cpd. DDD)

4-(Quinolin-4-ylamino)benzoic acid (Cpd A). To a mixture of4-chloroquinoline (0.5 g, 3.06 mmol) and 4-aminobenzoic acid (0.419 g,3.06 mmol) in anhydrous ^(i)PrOH 10 mL was added concentrated HCl 0.1 mLand heat to 150° C. in microwave for 1 h. The resulting reaction mixturewas cooled down to r.t. White precipitate formed and the crude productwas filtered and washed with ^(i)PrOH 3 mL×3. The crude product wasdried and analyzed with TLC. Single spot was detected and it wassufficiently pure (Cpd A) for the next step. Spectral Data: ¹H-NMR(DMSO-d₆/400 MHz): 15.05 (s, 1H), 11.24 (s, 1H), 8.91 (d, J=8.3 Hz, 1H),8.57 (d, J=7.0 Hz, 1H), 8.15 (d, J=7.4 Hz, 1H), 8.06 (m, 3H), 7.83 (m,1H), 7.78 (m, 2H), 7.03 (d, J=6.9 Hz, 1H). MS (ES+, m/z): 265.1 (M⁺+1,100.0).

4-(Quinolin-4-ylamino)benzoic acid (Cpd B). Cpd A (0.099 g, 0.294 mmol)in anhydrous thionyl chloride 3 mL was refluxed for 3 h, then evaporatedthe solution and got white crude product. The crude product, Cpd B, wasused for the next step directly.

Di-tert-butyl 4-(tosyloxy)pyrrolidine-1,2-dicarboxylate (Cpd C). To astirred solution of Boc-Hyp-OtBu (0.5 g, 1.74 mmol) in DCM (20 mL) wereadded pyridine (0.550 g, 6.96 mmol) and p-toluenesulfonyl chloride(0.663 g, 3.48 mmol) at r.t. After being stirred at r.t. for 24 h, thereaction mixture was quenched with 1M HCl and extracted with DCM. Theorganic layer was successively washed with 1M HCl, water, brine, driedover Na₂SO₄, and concentrated in vacuo. The crude product was purifiedby chromatography on silica gel and provided pure product, Cpd C (yellowsolid). Spectral Data: ¹H-NMR (CDCl₃/400 MHz): 7.82 (d, J=8.3 Hz, 2H),7.40 (d, J=8.3 Hz, 2H), 5.05 (m, 1H), 4.28 (m, 1H), 3.59 (m, 2H), 2.49(s, 3H), 2.57 (m, 1H), 2.13 (m, 1H), 1.47 (s, 9H), 1.45 (s, 9H). MS(ES+, m/z): 442.2 (M⁺+1, 30.0).

Di-tert-butyl 4-(4-aminophenylthio)pyrrolidine-1,2-dicarboxylate (CpdD). To a stirred solution of 4-aminothiophenol (0.072 g, 0.579 mmol) inanhydrous methanol (10 mL) were added sodium methoxide (0.039 g, 0.724mmol) and Cpd C (0.213 g, 0.482 mmol) at r.t. After being stirred for 16h at 80° C., the reaction mixture was quenched with saturated NH₄Cl andextracted with DCM. The organic layer was successively washed withwater, brine, dried over Na₂SO₄, and concentrated in vacuo. The productwas purified by chromatography (0% AE in Hexane) and provide purecompound, Cpd D. Spectral Data: ¹H-NMR (CDCl₃/400 MHz): 7.30 (d, J=8.3Hz, 2H), 6.64 (d, J=8.3 Hz, 2H), 4.20 (m, 1H), 3.90 (m, 1H), 3.40 (m,2H), 2.59 (m, 1H), 1.95 (m, 1H), 1.49 (s, 9H), 1.45 (s, 9H). MS (ES+,m/z): 395.2 (M⁺+1, 40.0).

Di-tert-butyl4-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)-pyrrolidine-1,2-dicarboxylate(Cpd F). Dissolved Cpd B (0.090 g, 0.253 mmol) and Cpd D (0.100 g, 0.253mmol) in anhydrous pyridine 5 mL and stirred at r.t. for overnight. Thenthe solvent was evaporated, and the resulting reaction mixture wasconcentrated and the pure product, Cpd F, was obtained as a colorlessoil by column chromatography using methanol/DCM, 0-5% ration solventsystem. Spectral Data: ¹H-NMR (CDCl₃/400 MHz): 9.06 (s, 1H), 8.68 (s,1H), 8.09 (m, 1H), 7.96 (m, 3H), 7.77 (d, J=8.3 Hz, 2H), 7.65 (m, 1H),7.42 (m, 1H), 7.36 (m, 2H), 7.25 (d, J=8.3 Hz, 2H), 6.76 (m, 1H), 4.21(m, 1H), 3.84 (m, 1H), 3.58 (m, 1H), 3.28 (m, 1H), 2.66 (m, 1H), 1.95(m, 1H), 1.49 (s, 9H), 1.45 (s, 9H). MS (ES+, m/z): 641.3 (M⁺+1, 100.0).

4-(4-(4-(Quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine-2-carboxylicacid (Cpd DDD). To a solution of Cpd E (0.020 g, 0.032 mmol) inanhydrous DCM 4 mL was added 0.5 mL 1,3-dimethoxybenzene and 2 mL of TFAat 0° C. The resulting reaction mixture was stirred at r.t. for 2 h. Thesolvent was evaporated and neutralized with NH₃ in methanol, thenacidified with HCl in dioxane. The crude product was purified withpreparative TLC by using 20% methanol in DCM. Cpd DDD was obtained inwhite solid. Spectral Data: ¹H-NMR (CD₃OD/400 MHz): 8.64 (d, J=8.4 Hz,1H), 8.47 (d, J=6.6 Hz, 1H), 8.13 (d, J=8.4 Hz, 2H), 8.03 (m, 2H), 7.84(m, 3H), 7.67 (d, J=8.3 Hz, 2H), 7.52 (d, J=8.3 Hz, 2H), 7.14 (d, J=8.3Hz, 1H), 4.10 (t, J=7.8 Hz, 1H), 3.92 (t, J=7.8 Hz, 1H), 3.62 (m, 2H),2.74 (m, 1H), 2.11 (m, 1H). MS (ES+, m/z): 485.1 (M⁺+1, 50.0).

Example EEE Preparation of3-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine (Cpd. EEE)

Tert-butyl 3-(4-aminophenylthio)pyrrolidine-1-carboxylate (Cpd F). To astirred solution of 4-aminothiphenol (0.220 g, 1.757 mmol) in methanol(10 mL) were added sodium methoxide (0.119 g, 2.197 mmol) andN-Boc-3-MsO-pyrrolidine (0.389 g, 1.464 mmol) at r.t. Then refluxed for1 d. After cooled to r.t., the reaction mixture was quenched withsaturated NH₄Cl and extracted with DCM. The organic layer wassuccessively washed with water, brine, dried over Na₂SO₄, andconcentrated in vacuo. FC with AE/HE 10-30% provided pure product, CpdF. Spectral Data: ¹H-NMR (CDCl₃/400 MHz): 7.30 (d, J=6.3 Hz, 2H), 6.64(d, J=6.3 Hz, 2H), 3.82 (br, 2H), 3.52 (m, 2H), 3.34 (m, 2H), 2.13 (m,2H), 1.88 (m, 1H), 1.49 (s, 9H). MS (ES+, m/z): 295.2 (M⁺+1, 40.0).

Tert-butyl4-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)-pyrrolidine-carboxylate(Cpd G). Dissolved Cpd B (0.181 g, 0.509 mmol) and Cpd. F (0.150 g,0.509 mmol) in anhydrous pyridine 5 mL and stirred at r.t. forovernight. Then the solvent was evaporated, and the crude product waspurified by methanol and DCM mixture 5-10% to yield Cpd G. Product,white solid. Spectral Data: ¹H-NMR (CDCl₃/400 MHz): 8.72 (d, J=5.1 Hz,1H), 8.13 (d, J=9.5 Hz, 1H), 7.99 (m, 3H), 7.65 (m, 3H), 7.48 (m, 2H),7.36 (d, J=8.3 Hz, 2H), 7.27 (d, J=8.3 Hz, 2H), 6.95 (br, 1H), 3.69 (m,1H), 3.56 (m, 1H), 3.37 (m, 2H), 2.19 (m, 1H), 1.93 (m, 1H), 1.49 (s,9H). MS (ES+, m/z): 514.2 (M⁺+1, 100.0).

3-(4-(4-(Quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine (Cpd EEE).To the solution of Cpd G (0.100 g, 0.185 mmol) in 5 mL anhydrous DCM wasadded 0.5 mL 1,3-dimethoxybenzene and 2 mL TFA at 0° C. The reactionmixture was stirred at r.t. for overnight. Then the solvent wasevaporated, and the crude product was neutralized with NH₃ in methanol.After removed the solvent, the reaction mixture was acidified with HClin dioxane. The crude product was purified by methanol and DCM mixture20% on preparative TLC to yield Cpd EEE. Spectral Data: ¹H-NMR(CD₃OD/400 MHz): 8.47 (m, 2H), 8.08 (d, J=9.5 Hz, 2H), 7.92 (m, 2H),7.72 (m, 2H), 7.58 (m, 1H), 7.56 (d, J=8.3 Hz, 2H), 7.46 (d, J=8.3 Hz,2H), 7.15 (d, J=8.3 Hz, 1H), 4.40 (m, 1H), 3.56 (m, 1H), 3.32 (m, 2H),2.42 (m, 1H), 2.02 (m, 1H). MS (ES+, m/z): 441.2 (M⁺, 60.0).

Example FFF Preparation ofN-(4-(piperidin-4-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide (Cpd.FFF)

Tert-butyl 4-(tosyloxy)piperidine-1-carboxylate (Cpd A). To a cooled inan ice bath solution of 1-Boc-4-hydroxypiperidine (2.013 g, 10.0 mmol)in dichloromethane (25 mL) pyridine (4 mL, 50 mmol), followed by asolution of p-tosyl chloride (3.810 g, 20.0 mmol) in dichloromethane (25mL) was added. The solution was stirred for 5 days. Colorless solutionwas diluted with dichloro-methane, washed 2× with 1M hydrochloric acid,with brine, 3× with 1M sodium hydroxide, with brine and dried withsodium sulfate. Solvent was removed under reduced pressure to give 5.25g of almost colorless oil. It was treated with ˜50 mL ofhexanes—crystallization started. Crystals were filtered off, washed 3×with hexanes and dried in vacuo to give Cpd A as white crystals. Purity(HPLC): 100%. It was immediately used for the next step.

Tert-butyl 4-(4-aminophenylthio)piperidine-1-carboxylate (Cpd. B). To astirred solution of Cpd A (1.066 g, 3.0 mmol) in DMF (15 mL) potassiumcarbonate (2.488 g, 18.0 mmol, 6 eq.), followed by a solution of4-aminothiophenol (0.789 g, 6.3 mmol, 2.1 eq.) in DMF (15 mL) was added.The suspension was stirred and heated at 70° C. (bath temperature)overnight. The mixture was partitioned between water and ethyl acetate.Aqueous layer was additionally extracted 2× with ethyl acetate. Extractswere combined and washed 2× with 1M sodium hydroxide then 2× with brine.The solution was dried over sodium sulfate and the solvent was removedunder reduced pressure to yield 1.36 g of pale brown thick oil. It waspurified by MPLC (24 g silica column, gradient 0-30% AcOEt in hexanes)to give a brown oil which solidified in vacuo to give Cpd B (0.730 g,79%). Purity (HPLC): 98%. MS (ES+): calc. for C16H24N2O2SNa (MNa+)331.1, found 331.3

4-(quinolin-4-ylamino)benzoic acid hydrochloride (Cpd C). To asuspension of 4-chloroquinoline (0.500 g, 3.06 mmol) and 4-aminobenzoicacid (0.419 g, 3.06 mmol) in 2-propanol (10 mL) 3 drops of concentratedhydrochloric acid were added and mixture was microwave irradiated at150° C. for 1 hour. After cooling solid was filtered off, washed with2-propanol and dried in vacuo to give Cpd C as a yellow powder. Purity(HPLC): 100%. 1H NMR (DMSO-d6): 7.05 (d, J=6.9 Hz, 1H), 7.64-7.67 (m,2H), 7.80-7.85 (m, 1H), 8.02-8.17 (m, 5H), 8.59 (d, J=7.2 Hz, 1H), 8.92(d, J=8.1 Hz, 1H), 11.26 (s, 1H). MS (ES+): calc. for C16H₁₃N₂O₂ (MH+)265.1, found 265.1.

Tert-butyl4-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)-piperidine-1-carboxylate(Cpd D). Solution of Cpd C (0.256 g, 0.85 mmol, 1.1 eq.) in thionylchloride (5 mL) was refluxed for 3 hours. Excess of thionyl chloride wasremoved under reduced pressure and residual yellow acid chloride wasdissolved in pyridine (5 mL). Solution of Cpd B (0.239 g, 0.78 mmol) inpyridine (5 mL) was added and red solution was stirred overnight at roomtemperature. The mixture was partitioned between ethyl acetate and 1Msodium hydroxide. Aqueous layer was additionally extracted 2× with ethylacetate. The extracts were combined, washed 2× with 1M sodium hydroxidethen with brine and dried over sodium sulfate. The solvent was removedunder reduced pressure and residue was co-evaporated with toluene toremove the remaining pyridine to give 0.458 g of a red solid. It wastriturated with DCM to give Cpd D as beige powder (0.235 g, 55%). Purity(HPLC): 88%. 1H NMR (DMSO-d6): 1.39 (s, 9H), 1.25-1.45 (m, 2H),1.80-1.92 (m, 2H), 2.70-3.00 (m, 1H), 3.25-3.40 (m, 2H), 3.78-3.88 (m,2H), 7.23 (d, J=4.5 Hz, 1H), 7.41-7.63 (m, 5H), 7.72-7.84 (m, 3H),7.92-8.05 (m, 3H), 8.39 (d, J=8.1 Hz, 1H), 8.59 (d, J=4.5 Hz, 1H), 9.30(bs, 1H), 10.27 (s, 1H).

N-(4-(piperidin-4-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide (CpdFFF). Suspension of Cpd D (0.235 g, 0.424 mmol) in a mixture of1,3-dimethoxybenzene (2 mL) and dichloromethane (5 mL) was cooled downin an ice bath and trifluoroacetic acid (3 mL) was added in one portion.Solids dissolved immediately and in ˜5 min. solution turned green. Bathwas removed and solution was left at room temperature overnight.Solvents from brown solution were removed under reduced pressure,residual oil was partitioned between ethyl acetate and diluted (1:2)concentrated hydrochloric acid. Aqueous layer was additionally extracted2× with ethyl acetate, basified with conc. sodium hydroxide andextracted 3× with ethyl acetate. Extracts were combined, washed withbrine, dried with sodium sulfate and solvent was removed under reducedpressure to give almost white solid of Cpd FFF. Purity (HPLC): 97%. 1HNMR (DMSO-d6): 1.25-1.45 (m, 2H), 1.72-1.92 (m, 2H), 2.76-3.44 (m, 5H),7.22 (d, J=5.1 Hz, 1H), 7.30-8.05 (m, 11H), 8.38 (d, J=8.7 Hz, 1H), 8.57(d, J=5.1 Hz, 1H), 9.25 (bs, 1H), 10.21 (s, 1H). MS (ES+): calc. forC27H27N₄OS (MH+) 455.2, found 455.4.

Example GGG Preparation ofN-(4-(piperidin-3-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide (Cpd.GGG)

Tert-butyl 3-(tosyloxy)piperidine-1-carboxylate (Cpd A). To a cooled inan ice bath solution of 1-Boc-3-hydroxypiperidine (1.006 g, 5.0 mmol) indichloromethane (15 mL) pyridine (2 mL, 25 mmol), followed by a solutionof p-tosyl chloride (1.906 g, 10.0 mmol) in dichloromethane (10 mL) wasadded. The solution was stirred for 5 days. Colorless solution wasdiluted with dichloromethane, washed 2× with 1M hydrochloric acid, withbrine, 3× with 1M sodium hydroxide, with brine and dried with sodiumsulfate. Solvent was removed under reduced pressure to give 2.56 g ofalmost colorless oil. It was purified by MPLC (24 g silica column,gradient 0-30% AcOEt in hexanes) to give Cpd A as a colorless thick oil.It was immediately used for the next step.

Tert-butyl 3-(4-aminophenylthio)piperidine-1-carboxylate (Cpd B). To astirred solution of Cpd A (1.487 g, 4.18 mmol) in DMF (20 mL) potassiumcarbonate (3.47 g, 25.1 mmol, 6 eq.), followed by a solution of4-aminothiophenol (1.100 g, 8.79 mmol, 1.1 eq.) in DMF (10 mL) wasadded. The suspension was stirred and heated at 70° C. (bathtemperature) overnight. The mixture was partitioned between water andethyl acetate. Aqueous layer was additionally extracted 2× with ethylacetate. Extracts were combined and washed 2× with 1M sodium hydroxidethen 2× with brine. The solution was dried over sodium sulfate and thesolvent was removed under reduced pressure to yield 1.452 g of yellowoil. It was purified by MPLC (24 g silica column, gradient 0-30% AcOEtin hexanes) to give Cpd B as an off white solid. Purity (HPLC): 100%. 1HNMR (CDCl3): 1.42 (s, 9H), 1.60-1.80 (m, 1H), 2.00-2.10 (m, 1H),2.70-2.92 (m, 3H), 3.70-4.30 (m, 4H), 6.64 (d, J=8.4 Hz, 2H), 7.30 (d,J=8.1 Hz, 2H).

4-(quinolin-4-ylamino)benzoic acid hydrochloride (Cpd C). To asuspension of 4-chloroquinoline (0.500 g, 3.06 mmol) and 4-aminobenzoicacid (0.419 g, 3.06 mmol) in 2-propanol (10 mL) 3 drops of concentratedhydrochloric acid were added and mixture was microwave irradiated at150° C. for 1 hour. After cooling solid was filtered off, washed with2-propanol and dried in vacuo to give Cpd C as a yellow powder. Purity(HPLC): 100%. 1H NMR (DMSO-d6): 7.05 (d, J=6.9 Hz, 1H), 7.64-7.67 (m,2H), 7.80-7.85 (m, 1H), 8.02-8.17 (m, 5H), 8.59 (d, J=7.2 Hz, 1H), 8.92(d, J=8.1 Hz, 1H), 11.26 (s, 1H). MS (ES+): calc. for C16H13N₂O₂ (MH⁺)265.1, found 265.1.

Tert-butyl3-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)-piperidine-1-carboxylate(Cpd D). Solution of Cpd C (0.331 g, 1.1 mmol, 1.1 eq.) in thionylchloride (5 mL) was refluxed for 3 hours. Excess of thionyl chloride wasremoved under reduced pressure and residual yellow acid chloride wasdissolved in pyridine (5 mL). Solution of Cpd B (0.308 g, 1.0 mmol) inpyridine (5 mL) was added and red solution was stirred overnight at roomtemperature. The mixture was partitioned between ethyl acetate and 1Msodium hydroxide. Aqueous layer was additionally extracted 2× with ethylacetate. The extracts were combined, washed 2× with 1M sodium hydroxidethen with brine and dried over sodium sulfate. The solvent was removedunder reduced pressure and residue was co-evaporated with toluene toremove the remaining pyridine to give 0.541 g of a yellow solid. It wasdissolved in small amount of boiling ethyl acetate and diluted with˜equal volume of hexanes. After cooling precipitate was filtered off,washed with AcOEt-hexanes (1:1), with hexanes and dried in vacuo to giveCpd D as a pale yellow powder. Purity (HPLC): 96%.

N-(4-(piperidin-3-ylthio)phenyl)-4-(quinolin-4-ylamino)benzamide (CpdGGG). Suspension of Cpd D (0.314 g, 0.566 mmol) in a mixture of1,3-dimethoxybenzene (2 mL) and dichloromethane (5 mL) was cooled downin an ice bath and trifluoroacetic acid (3 mL) was added in one portion.Solids dissolved immediately and in ˜5 min. solution turned green. Bathwas removed and solution was left at room temperature overnight.Solvents from brown solution were removed under reduced pressure,residual oil was partitioned between ethyl acetate and diluted (1:2)concentrated hydrochloric acid. Aqueous layer was additionally extracted2× with ethyl acetate, basified with conc. sodium hydroxide andextracted 3× with ethyl acetate. Extracts were combined, washed withbrine, dried with sodium sulfate and solvent was removed under reducedpressure to give almost white solid (0.246 g, 96%). It was trituratedwith ethyl acetate to give Cpd GGG as a pale yellow solid. Purity(HPLC): 97%. 1H NMR (DMSO-d6): 1.30-1.65 (m, 2H), 1.65-1.80 (m, 1H),1.95-2.10 (m, 1H), 2.50-2.65 (m, 2H), 2.90-3.05 (m, 1H), 3.05-3.30 (m,2H), 7.23 (d, J=5.4 Hz, 1H), 7.42-7.62 (m, 5H), 7.23-7.84 (m, 3H),7.93-8.04 (m, 3H), 8.41 (d, J=8.4 Hz, 1H), 8.59 (d, J=5.4 Hz, 1H), 9.32(bs, 1H), 10.29 (s, 1H). MS (ES+): calc. for C27H27N4OS (MH+) 455.2,found 455.3.

Example HHH Biological Activity of Compounds of the Invention

Test for the selective depletion of DNMT1 by 4-anilinoquinolines wasperformed with highly specific antibodies against all three enzymes, asdescribed by Ghoshal et al. [Mol. Cell. Biol. 2005, 11, 4727-41.]. ForDNMT1, commercially available antibodies from Santa Cruz or New EnglandBiolabs were used. Antibodies against DNMT3a and 3b with high titre,which did not cross-react with each other in Western blot orimmunoprecipitation analysis, were prepared fresh. Protein extracts(equivalent to 100 μg) were isolated from HCT116 cells (colon cancercells that express all three DNMTs at a relatively high level) that havebeen treated with 4-anilinoquinolines at concentrations 5, 10 and 100μM. As shown in Table 3, of the compounds screened, nine compounds at100 μM, thirteen at 10 μM, and eleven at 5 μM were able to induce DNMT1degradation greater than 60% (DNMT1 level less than 40%). Four compounds(Cpd. E, Cpd. EEE2, Cpd. II, and Cpd. FF1) were able to induce DNMT1degradation greater than 94% (DNMT1 level less than 6%) at 10 μM,exhibiting a level of potency on the order of that achieved bydecitabine.

The demethylating activity of non-bis-quaternary 4-anilinoquinolineswere tested in a cell-based GFP (green fluorescent protein) assay. Thisassay has a GFP gene regulated by the CMV promoter and is sensitive tothe methylation of CpG sites within the promoter. A decrease inmethylation resulting from exposure to a methylation inhibitor leads toGFP expression and is readily scored. Specifically, the CMV-EE210 cellline containing the epigenetically silenced GFP transgene was used toassay for reactivation of GFP expression by flow cytometry. CMV-EE210was made by transfecting NIH 3T3 cells with the pTR-UF/UF1/UF2 plasmid(Zolotuhin et al., 1996), which is comprised of pBS(+) (Stratagene,Inc.) containing a cytomegalovirus (CMV) promoter driving a humanizedGFP gene adapted for expression in mammalian cells. After transfection,high-level GFP expressing cells were initially selected by FACS analysisand sorting using a MoFlo cytometer (Cytomation, Inc.). Decitabine,potent inhibitor of mammalian DNMT1, was used as a positive control. Toscreen for reactivation of CMV-EE210, decitabine (at 1 μM) or a testcompound (at a concentration of 30-50 μM) was added to complete medium(phenol red free DMEM (Gibco, Life Technologies) supplemented with 10%fetal bovine serum (Hyclone)). Cells were then seeded to 30% confluence(˜5000 cell/well) in 96 well plate containing the test compounds andgrown for three days in at 37° C. in 5% CO₂. The plates were examinedunder a fluorescent microscope using a 450-490 excitation filter (13filter cube, Leica, Deerfield Ill.). Wells were scored g1 positive if10% of viable cells express GFP, g2 positive if 30% of viable cellsexpress GFP and g3 if greater than 75% of the viable cells express GFP.GFP IC 50 is the concentration of an inhibitor that (like an IC 50) isthe dose at which the GFP expression level goes from g3 to g1/2. Asshown in Table 3, of the compounds tested, six compounds (Cpd. O, Cpd.BB, Cpd. P, Cpd. Q, Cpd. Z, and Cpd. CC) reactivated transcription ofthe GFP gene at greater than 75% level. In addition, they are less thanone-half as toxic as decitabine.

TABLE 3 SUMMARY OF DEMETHYLATING ACTIVITY AND INDUCTION OF DNMT1DEGRADATION OF SELECT COMPOUNDS OF THE PRESENT INVENTION GFP % DNMT1Expres- Level sion IC₅₀ TD₅₀ Compound 5 μM 10 μM 100 μM Level (μM) (μM)Untreated 100 — — — — — Control Decitabine 0-5 — — g3 0.39 12.5 A 99 117119 ND — 12.5 V 112 110 124 ND — 12.5 K 98 92 37 ND — 12.5 L 144 145 128g2 50 50 O 84 150 142 g3 12.5 50 N 160 152 130 g2 100 50 F 145 210 155ND — 25 R 160 170 165 ND — 25 Y 93 63 77 g1 100 25 S 87 101 69 ND — 25AA 136 101 32 g2 100 25 J 101 89 59 ND — 12.5 T 64 69 85 ND — 25 D 131103 74 g1 — 12.5 C 31 33 34 g1 50 25 U 97 53 32 ND — 25 M 30 40 43 ND —25 X 49 98 73 g2 50 25 BB 106 76 32 g3 100 25 P 57 91 11 g3 50 25 H 13225 24 ND — 12.5 Q 219 93 10 g3 12.5 25 Z 83 115 87 g3 1.0 25 B 102 98 81ND — 25 DD 90 74 52 ND — 25 CC 65 81 26 g3 6.0 25 E 11 0.5 — g1 12.0 25I 27 80 48 ND — 25 G 95 95 100 — — EE 30 10 — — — W 45 75 — — — OO2 5095 — — — NN 70 70 — — — OO1 25 15 — — — QQ 60 50 — — — EEE2 3 0 — — — PP60 70 — — — GG1 25 20 — — — GG2 85 52 — — — JJ1 75 70 — — — JJ2 147 130— — — GG3 110 10 — — — II 25 0 — — — FF1 15 5 — — — FF2 85 83 — — — LL70 15 — — — KK 82 20 — — — HH — 90 — — — GG4 — 90 — — — GG5 — 90 — — —RR1 — 100 — — — RR2 — 100 — — — RR3 — 100 — — — ND-No activity detected;TD₅₀-Dose at which cells are > 50% viable

RT-PCR Assay for Gene Expression

RKO and HCT-116 cells were treated with Cpd. AAA, Cpd. BBB, and Cpd. CCCat varying concentrations. After a forty eight hour incubation cellswere harvested for RNA isolation using the Qiagen RNeasy Mini kit. RNAwas quantified using a spectrophotometer and 1 μg of RNA was used forcDNA synthesis using the Bio-Rad iScript cDNA Synthesis Kit. RTPCR wasperformed using SYBER GreenER qPCR SuperMix for iCycler according to themanufacturer's protocol. Primer sequences used are as follows p165′-atgtcctgccttttaacgta-3′ and 5′-gtgctcactccagaaaactc-3′, MLH-15′-tgaggaagggaacctgattg-3′ and 5′-tcttcgtcccaattcacctc-3′, p155′-caccatgaagcgaaacacag-3′ and 5′-tccatcggaagattcgtagc-3′, GAPDH5′-attgccctcaacgaccactt-3′ and 5′-ggtccaccaccctgttgc-3′, and B-actin5′-ctggaacggtgaaggtgaca-3′ and 5′-aagggacttcctgtaacaacgca-3′. Sampleswere analyzed on the iQ5 Multicolor Real-Time PCR Detection System fromBio-Rad. Data analysis was performed using the iQ5 Optical SystemSoftware version 2.0, Threshold Cycle (CT) and CT mean values weredetermined from this software. For each sample there are at least fourreactions, two for p16 and two for GAPDH the housekeeping control. Themean CT value from the GAPDH reactions was subtracted from each of thecorresponding p16 samples CT giving a value termed Delta CT. Then theDelta CT for the untreated p16 sample is subtracted from all treatedDelta CT values giving another value termed Delta Delta CT. The RelativeExpression value equals two to the power of the negative Delta Delta CTvalue (=2̂-Delta Delta CT). The duplicate samples are averaged togetherto get an average Relative Expression for each treatment and theStandard Error is calculated. Relative expression values for p15 andMLH-1 were calculated using the same method.

As seen in FIG. 1, Cpd. AAA, Cpd. BBB, and Cpd. CCC were able toincrease the RNA expression levels of p16. Cpd. AAA at the concentrationof 0.1 μM caused the greatest re-expression of p16 over no treatment.Because the higher levels of p16 seemed to have less effect in causingre-expression of p16 there may be some issues with drug solubility ortoxicity at the higher concentrations tested.

Cell-Based Growth Assays

Cell culture-based assays can be used to evaluate the ability ofcompounds of the invention to inhibit one or more cellular activities,such as cancer cell growth and/or survival. Numerous cancer cell linescan be obtained from the American Type Culture Collection (ATCC) andother sources. Briefly, cells are seeded into 96-well, tissue-culturetreated, opaque white plates (Thermo Electron, Vantaa, Finland), atbetween 5000 and 10000 cells per well, depending on the speed of cellproliferation, in 100 μl of appropriate growth medium (determined by theATCC). Cells are then exposed to the appropriate concentration of drugor an equal amount of DMSO (drug diluent) and allowed to grow in itspresence for 96 hours. Following this, 100 μl of Cell-Titer-Glo (CTG)reagent (Promega, Inc., Madison, Wis.) is added to each well. Plates arethen shaken for 2 minutes at room temperature to allow for cell lysisand incubated for 10 minutes at room temperature to stabilize theluminescent signal. Similar to the Kinase-Glo assay reagent fromPromega, this reagent contains both luciferase enzyme and its substrateluciferin. Luciferase, activated by ATP in the cell lysate, catalyzesthe conversion of luciferin to oxyluciferin, a reaction which produceslight. The amount of light produced is proportionate to the amount ofATP in the cell lysate, which is itself proportional to cell number andgives an index of cellular proliferation. IC₅₀ values for representativecompounds are set forth in Table 4 below.

TABLE 4 IC₅₀ Values CTG Cpd. Cpd. Cpd. Cell line AAA.Cl BBB.Cl CCC.ClHCT-116 6.15 μM 0.67 μM ND RKO 1.22 μM 0.74 μM 6.60 μM Panc-1 1.80 μM1.00 μM ND

DNMT1 Inhibition Assay

The DNMT1 Activity/Inhibition kit from Epigentek Inc. (Brooklyn, N.Y.)was used in this experiment (Catalog No. P-3006). In general terms, thisassay utilizes an active DNMT1 enzyme incubated withS-adenosylmethionine (SAM) in 96-well plates upon which unmethylated DNAhas been immobilized onto the surface of each well. After incubation thereaction wells are washed and probed with a primary anti-methylcytosineantibody, which will bind to methylated DNA. A secondary antibody isthen used to detect the primary antibody and creates a signal that isproportional to the amount of methylated DNA in the well. UninhibitedDNMT1 produces a well with high levels of methylated DNA (high signal),whereas inhibited DNMT1 produces a well with low levels of methylation(low signal). The final concentrations of reagents used in this DNMT1assay were as follows: 100 μM SAM and 2.2 μg/ml DNMT1 enzyme, withinhibitor concentrations typically ranging from 100 μM to 0.781 μM.

More specifically, test compounds were resuspended in 100% DMSO at 3 mMand serially diluted 1:2 in DMSO 8 times. A volume of 100 μl of 1× assaybuffer was combined with 12 μl of 1 mM SAM solution and 4 μl of 66.7μg/mL DNMT1 enzyme in the presence of 4 μl of test compound diluted inDMSO. Controls include enzyme only (no inhibitor, but containing 3.3%DMSO), no enzyme, and compounds having inhibitory activity. A volume of30 μl of the mixed solution was then added to the substrate-coated wellsin triplicate and incubated at 37° C. for 2 hours. Reaction wells arethen washed 3× with 150 μl of DNMT wash buffer. The primary antibody(anti-methylcytosine) was diluted 1:1000 in wash buffer, added to eachwell (50 μl) and incubated at room temperature for 1 hour. Reactionwells were washed again. The secondary antibody was diluted 1:1000 inwash buffer, added to each well (50 μl) and incubated at roomtemperature for 30 min. The wells were washed for the last time. Thedeveloping solution was added to each well (100 μl) and incubated atroom temperature for 4 minutes. The stop solution was added (50 μl) andthe plate was immediately read on a plate reader at 450 nm absorbance.Compounds DDD, EEE and GGG were tested by this assay and gave values of1.6 μM, 5.5 μM and 0.31 μM, respectively.

While this invention has been described with reference to certainembodiments and examples, it is to be appreciated that furthermodifications and variations can be made to embodiments and exampleswithout departing from the spirit or scope of the invention.

1. A compound selected from the group consisting of4-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine-2-carboxylicacid; 3-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine;N-(4-(piperidin-4-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide; andN-(4-(piperidin-3-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide, or aphysiologically acceptable salt thereof.
 2. The compound of claim 1,wherein the compound is4-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine-2-carboxylicacid, or a physiologically acceptable salt thereof.
 3. The compound ofclaim 1, wherein the compound is3-(4-(4-(quinolin-4-ylamino)benzamido)phenylthio)pyrrolidine, or aphysiologically acceptable salt thereof.
 4. The compound of claim 1,wherein the compound isN-(4-(piperidin-4-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide, or aphysiologically acceptable salt thereof.
 5. The compound of claim 1,wherein the compound isN-(4-(piperidin-3-ylthio)phenyl)-4-(quinolin-4-ylamino)-benzamide, or aphysiologically acceptable salt thereof.
 6. The compound of claim 1,wherein the physiologically acceptable salt is formed with an acidselected from the group consisting of hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, carboxylic, sulfonic, sulfo orphospho acids, acetic acid, propionic acid, glycolic acid, succinicacid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid,malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid,glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinicacid, isonicotinic acid, amino acid, glutamic acid, aspartic acid,phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid, andascorbic acid.
 7. The compound of claim 1, wherein the physiologicallyacceptable salt is a sodium, calcium, lithium, potassium, ammonium, ortrialkylammonium salt.
 8. A pharmaceutical composition comprising acompound of claim 1, or a physiologically acceptable salt thereof, and apharmaceutically-acceptable carrier.
 9. The pharmaceutical compositionof claim 8, wherein the pharmaceutical composition is in a solid form.10. The pharmaceutical composition of claim 8, wherein thepharmaceutical composition is in an oral dosage form.
 11. Thepharmaceutical composition of claim 8, wherein the pharmaceuticalcomposition is in an injectable dosage form.
 12. The pharmaceuticalcomposition of claim 8, wherein the pharmaceutical composition is in atopical dosage form.
 13. A method for inhibiting DNA methylation in acell, comprising contacting the cell with a compound of claim 1, or aphysiologically acceptable salt thereof, such that DNA methylationactivity of the cell is inhibited.
 14. A method for inhibiting DNAmethylation in a cell, comprising contacting the cell with a compound ofclaim 1, or a physiologically acceptable salt thereof, such that DNAmethyltransferase activity in the cell is inhibited.
 15. The method ofclaim 14, wherein the activity of DNA methyltransferase activity isinhibited via degradation of DNA methyltransferase DNMT1.
 16. The methodof claim 14, wherein the step of contacting includes contacting the cellwith a biologically effective amount of a compound of claim 1, or aphysiologically acceptable salt thereof, such that at least 50% of theactivity of DNA methyltransferase DNMT1 in the cell is inhibited. 17.The method of claim 14, wherein the step of contacting includescontacting the cell with a biologically effective amount of a compoundof claim 1, or a physiologically acceptable salt thereof, such that atleast 25% of the activity of DNA methyltransferase DNMT1 in the cell isinhibited.
 18. A method for restoring activity of a DNAmethylation-suppressed gene in a cell, comprising contacting a cell witha biologically effective amount of a compound of claim 1, or aphysiologically acceptable salt thereof, such that activity of the DNAmethylation-suppressed gene is elevated by at least 25% relative to thatin the absence of the compound or physiologically acceptable saltthereof.
 19. The method of claim 18, wherein the step of contactingincludes contacting the cell with a biologically effective amount of thecompound of claim 1, or a physiologically acceptable salt thereof, suchthat transcriptional activity or levels of transcript of theDNA-methylation-suppressed gene is elevated by at least 25%.
 20. Themethod of claim 20, wherein the DNA methylation-suppressed gene isselected from the group consisting of 14-3-3 Sigma, ABL1 (P1), ABO, APC,AR (Androgen Receptor), BLT1 (Leukotriene B4 Receptor), BRCA1, CALCA(Calcitonin), CASP8 (CASPASE 8), Caveolin 1, CD44, CFTR, COX2, CSPG2(Versican), CX26 (Connexin 26), Cyclin A1, DBCCR1, ECAD (E-cadherin),Endothelin Receptor B, EPHA3, EPO (Erythropoietin), ER (EstrogenReceptor), FHIT, GPC3 (Glypican 3), GST-pi, H19, H-Cadherin (CDH13),γ-globin, HIC1, hMLH1, HOXA5, IGF2 (Insulin-Like Growth Factor II),IGFBP7, IRF7, LKB1, LRP-2 (Megalin), MDGI (Mammary-derived growthinhibitor), MDR1, MDR3 (PGY3), MGMT (O6 methyl guanine methyltransferase), MUC2, MYOD1, N33, NEP (Neutral Endopeptidase 24.1)/CALLA,NIS (sodium-iodide symporter gene), P14/ARF, P15 (CDKN2B), P16 (CDKN2A),P27KIP1, p57 KIP2, PAX6, PgR (Progesterone Receptor), RAR-Beta2, RASSF1,RB1 (Retinoblastoma), TERT, TESTIN, TGFBRI, THBS1 (Thrombospondin-1),TIMP3, TLS3 (T-Plastin), Urokinase (uPA), VHL (Von-Hippell Lindau), WT1,and ZO2 (Zona Occludens 2).
 21. A method for treating a patientsuffering from a disease associated with aberrant DNA methylation,comprising administering to the patient the pharmaceutical compositionof claim
 8. 22. The method of claim 21, wherein the pharmaceuticalcomposition is administered orally, parenterally, topically,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery, subcutaneously, intraadiposally, intraarticularly, orintrathecally.
 23. The method of claim 21, wherein the pharmaceuticalcomposition is administered orally.
 24. The method of claim 21, furthercomprising administering to the patient a second therapeutic agent incombination with the pharmaceutical composition.
 25. The method of claim24, wherein the second therapeutic agent is decitabine or azacitidine.26. The method according to claim 24, wherein the second therapeuticagent is selected from the group consisting of histone deacylaseinhibitors, antibiotic agents, alkylating agents, retinoids, hormonalagents, plant-derived agents, biologic agents, interleukins,interferons, cytokines, immuno-modulating agents, and monoclonalantibodies.
 27. The method according to claim 26, wherein the histonedeacylase inhibitor is selected from the group consisting oftrichostatin A, suberoylanilide hydroxamic acid, oxamflatin, subericbishydroxamic acid, m-carboxy-cinnamic acid bishydroxamic acid,pyroxamide, trapoxin A, apicidin, depsipeptide,N-(2-amimophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide,butyric acid, phenylbutyrate and arginine butyrate.
 28. The methodaccording to claim 21, wherein the disease associated with aberrant DNAmethylation is selected from the group consisting of hematologicaldisorders, benign tumor and cancer.
 29. The method according to claim28, wherein the hematological disorder is selected from the groupconsisting of acute myeloid leukemia, acute promyelocytic leukemia,acute lymphoblastic leukemia, chronic myelogenous leukemia,myelodysplastic syndromes, and sickle cell anemia.
 30. The methodaccording to claim 28, wherein the cancer is selected from groupconsisting of breast cancer, skin cancer, bone cancer, prostate cancer,liver cancer, lung cancer, non-small cell lung cancer, brain cancer,cancer of the larynx, gall bladder, pancreas, rectum, parathyroid,thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi,and kidney cancer, basal cell carcinoma, squamous cell carcinoma of bothulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma,Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor,small-cell lung tumor, gallstones, islet cell tumor, primary braintumor, acute and chronic lymphocytic and granulocytic tumors, hairy-celltumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma,mucosal neuronms, intestinal ganglloneuromas, hyperplastic corneal nervetumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor,leiomyomater tumor, cervical dysplasia and in situ carcinoma,neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid,topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi'ssarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, and epidermoid carcinomas.